Gel Electrophoresis Size Marker

Gel Electrophoresis
Size Marker
Take the Pink Link!
www.
.com
Transfer
Membranes
“Thinkingwithoutknowledge
makeschancetheruler.”
Take the pink Link!
Take the pink Link!
www.
.com
www.
Take the Pink Link!
.com
www.
WernerKollath,Germanbacteriologist
Detergents
Kontaminationen
Nukleinsäuren
Immunoassay
Buffer
Probleme & praktische Lösungen
durch
.com
Gel Electrophoresis
Size Marker
Ever since the foundation of the firm, AppliChem has
investedextensivelyincommunicationandmarketing.The
freshandunusualappearanceattractedgreatattentionin
Take the Pink Link!
Take the Pink Link!
www.
Take the Pink Link!
.com
www.
Take the Pink Link!
.com
themarketfromtheverybeginning.Ourgrowthconfirms
www.
.com
www.
AgaroseElektrophorese
.com
AppliCations
AppliCa
therelevanceofthesemeasures.
Wichtiges
Wissenswertes
Wunderbares
aus Chemie & Biologie
Nr.1
Weofferourcustomersanextensivelibrary,withnumerous
Improving quality o
Nukleinsäure-Dekontamina
mit der ExitusPlus™-Tec tion
hnologie
Die moderne Gentechnik
zeigt, dass in vielen Fällen
schon freie DNA-Moleküle
für Infektionen, Rekombin
ationen oder biologische
Transformationen ausreiche
n
[1,2]. Zusätzlich werden
die Nachweisverfahren
für DNA-Moleküle immer
sensitiver. Daher wird die
Detektion von Kontaminationen
oder die Verhinderung
von Amplifikations-Artefakten
in der PCR für die Gentechn
ik, die Kriminalistik, die
Biomedizin und die Hygiene
immer wichtiger. Die vollständi
ge Dekontamination
von Geräten und Materialie
n von DNA-Molekülen wird
so zu einem entscheidenden
Faktor für die allgemeine
biologische Sicherheit.
Using ready-to-use ELISA
brochuresandapplicationswhoseusemakeseverydaylife
inthelaboratoryeasier.
Keywords
Take the Pink Link!
NukleinsäureDekontamination
Take the Pink Link!
.com
DNA-Degradationstest
Alles oder Nichts: Erstaunlich
e Erkenntnis
Das Mittel der Wahl zur Beseitigung von
Kontaminationen durch Nukleinsäuren
Kontamina
ist immer noch Chlorbleichlauge („bleach“)
– ein Mittel das alles zerstört, nicht nur
die Nukleinsäure. Dies hat uns veranlasst
in Kooperation mit multiBIND Biotech,
Köln, nach einer unschädlichen Alternative
zu suchen und die molekulare Wirkungsweise
sonstigen DNA-Dekontaminationsmittel
der auf dem Markt befindlichen
zu untersuchen.
suchen. Hierfür wurde unter sehr hoher
schuss) mit definierten DNA-Kontami
Belastung (großer DNA-Über
DNA-Übernationen die Eigenschaften der konventionelle
n Mittel verglichen. Zwei Probleme
werden offensichtlich: Erstens werden
durch die konventionellen Mittel in keinem
Fall die DNA-Moleküle effizient zerstört
und zweitens enthalten diese Mittel Komponenten
mit stark korrosiven oder giftigen Eigenschaften
für uns die Notwendigkeit der Neuentwicklun
. Als Fazit daraus hat sich
g einer effektiven Lösung zur DNA-Dekontam
DNA-ExitusPlus™ und Autoclave-Exit
ination ergeben, die wir hier als
usPlus™ vorstellen. Im Vergleich zu
den herkömmlichen Produkten wird
RNA schnell und effizient zerstört, ohne
DNA und
dass das Reagenz korrosive oder giftige
Eigenschaften aufweist.
Bei der DNA-Dekontamination unterscheidet
man nach der molekularen Wirkungsweise
der eingesetzten Mittel drei Grundprinzipien zur Zerstörung oder Inaktivierung
der genetischen Information: Modifika
Modifikation, Denaturierung und Degradation.
Je nach Zusammensetzung der Mittel
können diese drei Prinzipien einzeln
oder in Kombination angewandt werden.
Da nach den aktuellen Erkenntnissen
zum biologischen Risikopotenzial von
freien
DNA-Molekülen für eine wirklich sichere
DNA-Dekontamination die Zerlegung
dieser DNA-Moleküle in möglichst kleine
Fragmente die wirkungsvollste Methode
wurden die gängigen konventionelle
tionellenn Mittel mit unserer Neuentwicklun
ist,
g DNA-ExitusPlus™ im DNA-Degradat
glichen. Der DNA-Degradationstest erlaubt
ionstest ver
vereinen sensitiven, quantitativen Vergleich
der Geschwindigkeit des DNA-Abbaus
(Abb. 1 und 2).
Unerwarteter Weise haben wir festgestellt,
dass einige der bekannten kommerziellen
Mittel nur mit dem Prinzip der Modifikation oder Denaturierung der DNA-Moleküle
arbeiten. Eine Zerlegung der DNA-Stränge
genetische Informa
erfolgt dabei nicht, sondern die
Information, für die diese DNA-Stränge
kodieren, wird eigentlich nur maskiert.
der DNA-Moleküle durch Entfernung
Eine chemische Demaskierung
der blockierenden Gruppen würde die
genetische Information wieder lesbar
plifizierbar machen. Nach dem heutigen
und amWissensstand zur Gentechnik und der
Problematik der Neukombination von
trägern sind solche Mittel eigentlich nicht
Erbmehr zeitgemäß. Aber auch die Mittel,
die zu einer nachweisbaren Degradation
.com
PCR-Test
Autoklavieren von DNA
AppliCations
because after storage of some
Keywords
Immunoassays
Antibody Stabilisation
ELISA Plates
Cross-reactivity
Interfering effects
Dekontamination der Haut
und Hände von Nukleinsäure
n
AppliCations
No.6
Size-Exclusion Chromato
graphy
for purification of biomolec
ules
Freie Nukleinsäuren verursach
en als Kontaminationen
große Probleme im
Forschungs- und molekular
biologisch-analytischen oder
klinisch-diagnostischen
Labor. Durch die extrem hohe
Sensitivität von DNA-Nach
weistests, können kleinste
Verunreinigungen in PCR-Ansä
tzen zusätzliche Arbeit bedeuten
und im schlimmsten Fall Ergebnisse verfälsch
en. Mit Derma-ExitusPlus™
(HHDK) aus der Serie
von ExitusPlus™-Produkten
wird erstmals ein völlig
neuer Anwendungsbereich
erschlossen bzw. zusätzlich
e Kontaminationsquellen
ausgeschlossen.
Size-exclusion chromatog
raphy (SEC) is a popular method
to separate biomolecules
based on their size. Primarily,
it is applied to the separation
of biopolymers such as
proteins and nucleic acids,
i.e. water-soluble polymers.
This system is also called
gel
filtration, typically with beads
of dextran or agarose serving
as gel matrix. Smaller
molecules pass significant
ly slower through the column
than larger molecules. Not
to
be mixed up with gel electropho
resis, there are big difference
s in terms of the
separation principle. SEC
does not require electric current
and the sieving effect will
not separate small molecules
first.
SizeMarker•©2010AppliChem
ine der Hauptquellen für Kontamination
en mit Nukleinsäuren ist der Experimentato
r selbst. Die Nukleinsäuren stammen
B. aus Hautschuppen, Haaren und Speichel
oder von Mikroorganismen, die seine
Haut besiedeln oder z.B. beim Niesen
eigesetzt werden. Gelangen diese in
die PCR-Ansätze oder PCR-Reagenzi
en, können s
Keywords
It is indeed correct that smaller molecules
pass more slowly through t
new measur
days the plates d
Why is there such a great difference
in storage between h
The reason is that in professional ELISA
kit production the plates are not
easy to perform process has been an industry
standard for thirty years. Fo
coating stabiliser solution. It is just as
simple as a “second blocking step
lutions freely available in low volumes for
use in research lab until now. AppliC
in volumes starting as small as 50 ml, which
is called the AppliCoat Plate Stabi
to-use and has a great advantage compared
to almost any stabiliser used
antibodies and antigens than most other
products do. And there is a secon
Two benefits with one solution
When antibodies are coated onto ELISA
plates, most of the antibodies are not
into close contact to the plastics surface
of the ELISA plate, conformational ch
The result is that most antibodies coated
on a plate are unfolded or inactive. O
active and can bind to analytes and this
is greatly variable depending on the s
really differ from batch to batch or even
from well to well.
These differences from well to well can
affect the variability of an assay, bec
way of refolding antibodies and of preserving
antibodies from conformati
decrease such variabilities in assay performance.
This is a key benefit of Ap
coated proteins to refold and then to
preserve active conformation over a lo
antibody conformation of some of the
coated antibodies and 2. Preserving c
fits are used for production of high-quality
ELISA kits as well as in resear
Stabiliser the percentage of active antibodies
will still be in the range of 2–8
from well to well and from plate to plate
can be minimised in most assays
depend on the used antibodies, but when
ELISA are validated (e.g. according
Validation”, FDA, 2001) or according to
other validation strategies, the differe
The positive effects of AppliCoat Plate
Stabiliser are shown in Fig. 1. A sandwi
AppliChembringsadvantagesthroughknowledge.
Nr.5
characteristics of the
detection are not appropria
te. Ready-to-use EL
stored for two years at 4°C
without any probl
information provided by no other catalogue in the field,
isavailableinGermanandEnglish.
de ELISA is required b
completely different story.
For any
A comprehensive catalogue of products, with detailed
www.
kits from manufactu
times however, home-ma
the right antibodies or the
AppliCations
DNA-freie Reagenzien
und Mastermixe für die PCR
Nr.3
Der Anteil von molekular
biologischen Nachweismethoden
ist in den letzten Jahren
erheblich gestiegen, besonder
s in den Bereichen Qualitätsk
ontrolle, Forensik,
klinischer Forschung und
Diagnostik – insbesondere
der Infektionsdiagnostik
.
Gerade für diese Applikatio
nen werden hochsensitive
und gleichzeitig zuverläss
ige
PCR-Tests benötigt. Dafür
bietet AppliChem nun optimiert
e PCR-Kits an und widmet
sich explizit der Hintergru
ndproblematik, die durch
DNA belastete Reagenzie
n und
Arbeitsplätze entstehen kann.
contents
1 DNA marker
2
1.1 Tips on the use of DNA length markers 2
1.2 AppliChem DNA marker overview
4
1.3 DNA marker
5
2 Dyes for nucleic acids 16
2.1 Overview
16
2.2 Methylene blue
16
2.3 Ethidium bromide
18
3 Protein marker
19
3.1 AppliChem protein marker overview
19
3.2 Precision protein markers
19
3.3 Prestained protein markers
21
4 Dyes for protein gels
24
4.1 Overview
24
4.2 Coomassie stain
24
4.3 Proteo-Dye
26
© 2010 AppliChem • Size Marker
1
Tips on the use of DNA length markers
1.1
Correct storage
Deproteinised and lyophilised DNA samples are extremely stable (> 5 years). Problems do not usually occur unless DNA
markers in solution are stored (> 6 weeks) at room temperature, they become contaminated with bacteria, or are frequently ­thawed and refrozen (> 20 times). After dissolution of the DNA, we therefore recommend aliquoting of the DNA marker
in ready-to-use aliquots for storage at -20°C (for 2-4 years). At +4°C, markers in solution are stable for several weeks
or even months. Here, however, there is the risk of bacterial or nuclease contamination. This can be prevented by storage
at -20°C.
End-labeling
Thanks to their deproteinised and lyophilised form, these DNA markers are suitable for universal use for end-labeling with
modified nucleotides. Labeling in four positions via the terminal EcoR I generated recessed ends is possible, especially with
the DNA ­ladder 100 bp (A3470) and the DNA Ladder Mix 100-5000 (A3660). For the labeling of the DNA, the product is
simply dissolved in TE buffer or bidistilled water.
DNA staining with methylene blue
Occasionally, ethidium bromide is not suitable for staining DNA. An alternative is methylene blue. In such cases, however, it
must be kept in mind that the mass of DNA used must be increased by about 30 % and that about 1.5 h more must be ­planned
for destaining steps. With a methylene blue concentrate supplied by AppliChem, you achieve very good results, even for small
fragments of about 200 bp.
Mass calculations for single fragments
Using DNA markers of a defined origin, the calculation of the mass of single fragments is relatively easy. The amount loaded
per lane, e.g. 1 µg/10 µl, is divided by the number of base pairs of the DNA used and is multiplied by the fragment size. For
example: the 267 bp fragment of the pBR322 Hae III marker with a loading amount of 1 µg: 1 µg : 4361 bp (pBR322) =
0.229 ng/base pair x 267 bp of the fragment = 61.2 ng/267 fragment.
Assuming comparable staining (saturation with dye), the mass of unknown fragments can be determined in this way. If
­required, concentration gradients can be loaded.
Less is often more
The different gel formats for agarose and polyacrylamide gel electrophoresis and the varying sensitivity of staining or detection mean that it is only possible to give an approximation of the recommended DNA amount to be loaded. Most DNA markers show the best separation with loading amounts of 0.5-1 µg on ­agarose gels. The general rule is: the lower the number
of marker bands, the smaller the total amount needed. A low loading amount is also of advantage with short migration
distances or very ­sensitive detection. When using polyacrylamide gels, generally only about 1/5-1/2 the amount required for
agarose gels is necessary. For DNA markers with large fragments and a high mass (e.g. equimolar mixtures or lambda DNA
markers), depending on the agarose concentration, a minimum separation distance is required to separate or completely
distinguish between the dominant upper bands in terms of width. This separation distance can often be reduced by 10-15
% by ­reducing the marker amount. By using equalized markers, in addition to a marked reduction of the marker amount,
the ­separation distance can be reduced by a further 10-15 %, from e.g. 70 to 50 mm, on a standard format gel, to obtain
the best results.
i n t r o
2
Size Marker • © 2010 AppliChem
How to achieve results quickly
Especially deproteinised DNA length markers can be separated very well using high voltages, e.g. within 20 minutes with a
migration distance of 70 mm on a 1.2 % agarose gel at 120 Volt. This can only be realized, however, with a high quality gel
­migration buffer (the water quality must be taken into account!) and an adequate amount of buffer (400-600 ml). Poor
buffer quality results in overheating of the agarose gel at higher voltages and this therefore causes problems. A high buffer
volume (the buffer can usually be reused 4-6 times in one week without problems) leads the heat off, provided the gel
chamber for submersion is of adequate size.
Staining front too intense?
In some cases, users who regularly perform assays with our products sometimes comment that the dye concentration in the
gel loading buffer we supply is too high (only lyophilised markers). If this is the case, simply dissolve the DNA length ­marker
in double concentration in the gel loading buffer and further dilute it with 1/2 volume TE buffer. Even when using a 50 %
solution, there are no problems with the resulting 7 % glycerol concentration to increase the solution density.
Plausibility of the results and ion concentrations:
Non-plausible results (e.g. unexpected fragment runs at 500 bp instead of 350 bp) may occur under relatively extreme
­conditions (separation of restriction or PCR samples with a very high salt content > 150 mM) or at very high voltages with
relatively short separation distances. To identify errors, 1/10 volume restriction buffer can be added to the aliquot of the DNA
marker, for example, or the restriction sample can be appropriately diluted with gel loading buffer (usually 1/2 volume). In
many cases, lowering the voltage during separation is also helpful. The accurate determination of the size of fragments or
PCR ­products may also be impaired, when salt fronts combined with local problems of leading off heat with conducting away
heat and very high ­electrophoresis voltages, result in partial denaturation of the DNA fragments. These are conditions that
theoretically may also occur when processing samples from Maxam-Gilbert sequencing in combination with sample buffers
containing formamide.
d u c t i o n
© 2010 AppliChem • Size Marker
3
Prod. No.
A3470
A5191
A3302
A5216
A3660
A3982
A8640
A2667
A5207
A6430
A7215
A7222
A4406
A5229
A6927
A5194
A4412
A5220
A5589
A5223
A5235
A3996
DNA Marker
Bands
DNALadder100bp(lyophilised)
11
DNALadder100bp
10
DNALadder100bpequalized(lyophilised)
11
DNALadder100bpplus
11
DNALadderMix100-5000(lyophilised)
17
DNALadder250bp(lyophilised)
16
DNALadder250bpplus(lyophilised)
15
DNALadder1kb(lyophilised)
11
DNALadder1kb
13
DNALadder1kbconcatamer(lyophlised)
>25
DNAMarkerquick-run(lyophylised)
5
DNAMarkerquick-runextended(lyophylised)
9
DNAMarkerpBR322-HaeIII(lyophilised)
22
DNAMarkerpBR322-HaeIII
22
DNAMarkerpBR328Mix(lyophilised)
12
DNAMarkerPhageLambdaStyI
11
DNAMarkerPhageLambdaBstEII(lyophilised)
BstEII(lyophilised)
Bst
14
DNAMarkerPhageLambdaBstEII
BstEII
Bst
14
DNAMarkerPhageLambdaHindIII(lyophilised)
HindIII(lyophilised)
Hind
8
DNAMarkerPhageLambdaHindIII
HindIII
Hind
8
DNAMarkerpUC19-MspI
12
DNAMarkerpUC19-MspI(lyophilised)
12
Fragment Sizes [bp]
1000
900
800
700
1000
900
800
700
1000
900
800
700
1500
1000
900
800
5000
4000
3000
2500
8000
6000
5000
4000
12000 10000 8000
6000
10000
8000
6000
5000
10000
8000
6000
5000
1000 bpsteps(1000-approx.25000)
2500
2000
1500
1000
6000
4000 25000
2000
587
540
502
458
587
540
502
458
2176
1766
1230
1033
19329
7743
6223
4254
8454
7242
6369
5686
8454
7242
6369
5686
23130
9416
6557
4361
23130
9400
6557
4361
501
489
404
331
501
489
404
331
600
600
600
700
2000
3000
5000
4000
4000
500
500
500
600
1500
2750
4000
3000
3000
400
400
400
500
1000
2500
3000
2500
2500
500
1500
434
434
653
3472
4822
4822
2322
2322
242
242
1000
267
267
517
2690
4324
4324
2027
2027
190
190
500
234
234
453
1882
3675
3675
564
564
147
147
Lyophilised markers
Starting material
TheDNAinourlyophilisedlengthmarkersisamplifiedinlow-nucleasehostbacteria,restrictedwithqualityenzymes,and
isdeproteinised.Thisensuresthattheycontainhigh-quality,low-proteinornuclease-freeproductswithverygoodmigration
propertiesonagarose,agarandpolyacrylamidegels.
Quality control
Numerous gel separations of undigested plasmids and digested fragments form part of the manufacturing process. The
fragmentmassisdeterminedusingtwocalibratedphotometersviaconversion(1OD260nm=50µgdsDNA)andisaliquoted
at100%(+2%).EachbatchofDNAandgelloadingbufferissubjecttoafinalqualitycheckusinggelelectrophoresis.
Stability
Lyophilisation of the DNA markers results in very stable products, without any significant impairment of quality at
room temperature after transport (even at the height of summer, > 35°C for several days) or after long storage times
(>4yearsat-20°C).
Resuspension
Beforeuse,lyophilisedDNAmarkerscanbedissolvedingelloadingbufferoroptionalforend-labelinginsterile,bidistilled
water. The lyophilised form allows you the highest degree of flexibility (labeling, concentration, intensity of staining
bands).
Legal note
TheDNAladdersweremanufacturedbydigestionofplasmidswithEcoR I.Theplasmidsarelegallyprotected.Toproduce
copiesapplyforapermissionfromthemanufacturer.
4
SizeMarker•©2010AppliChem
300
300
300
400
900
2250
2000
2000
2000
200
200
200
300
800
2000
1750
1500
1500
150
100
150
200
700
1750
1500
1000
1000
100
100
100
600
1500
1250
500
750
500
1250
1000
500
400
1000
750
250
300
750
500
200
500
250
150
250
100
200
213
213
394
1489
2323
2323
125
125
111
111
100
192
192
298
925
1929
1929
110
110
184
184
234
421
1371
1371
67
67
124
124
220
74
1264
1264
34
34
123
123
154
702
702
26
26
104
104
89
89
80
80
64
64
224
224
117
117
1.2
57
57
51
51
21
21
18
18
11
11
8
8
selection guide
DNA marker
DNA Ladder 100 bp (lyophilised)
A3470
DNAsizestandardformedium-sizedfragmentsandPCRproducts
Description
Thefragmentsofthissizemarkeroccurinequimolarproportions,i.e.alsoallpossiblemarking
positionsontheterminalEcoRIgeneratedrecessedends.Thebandmassincreaseswithfragment
length.TheDNAisdeproteinizedandlyophilized.Thismarkerisparticularlysuitableforcomparisonswithmedium-sizedDNAfragmentsandPCRproducts.The500basepairbandmasshas
beendoubledforeasierorientationon1.5-2.0agarosegels.Thesizeandmassofplasmidin-sertions or PCR products can be determined precisely in the range of up to 1000 base pairs at
intervals of 100 base pairs. An additional band with 150 base pairs has been included for the
determinationofsmallerPCRproducts.Thebestresultsareachievedafteramigrationdistanceof
approx. 70-80 mm on agarose gels. Each lane of a normal sized agarose gel (approx. 80 ml)
shouldbeloadedwithapprox.0.4-0.8µgofmarker.Loadingbufferissuppliedseparately.
Number of bands
11
Fragment sizes (bp) 1000,900,800,700,600,500(x2),400,300,200,150,100
Supplied with 1 ml 10mMTris·HCl(pH7.5);5mMsodiumacetate;2mMEDTA;10%glycerol;
loading buffer (1X) 0.02%bromophenolblue;0.015%xylenecyanol
Storage
-20°C
Loading volume
0.4-0.8µgperlane
Package size
A3470,0050
50µg
1.3
©2010AppliChem•SizeMarker
5
The quality of lyophilised DNA sizing standards differs considerably from products offered by most other manu­
facturers thanks to the elaborate manufacturing method, which also fulfils the highest ­quality requirements!
The digested DNA is not simply precipitated and resuspended, but also extracted in phenol. This guarantees the
extremely long shelf-life without any loss of quality. Not only the prices should therefore be compared.
DNA Ladder 100 bp
A5191
Description
1 00 bp DNA ladder is ideal for determining the size of double-stranded DNA from 100 to 1 000
base pairs. The 100 bp and 500 bp fragments are present at increased intensity to allow easy
identification. All fragments are blunt-ended.
Number of bands
10
Fragment sizes (bp) 1000, 900, 800, 700, 600, 500 x 2, 400, 300, 200, 100 x 2
Storage buffer
10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
Concentration
0.2 mg/ml
Storage
-20°C
Loading volume
1 µg per lane
Package sizes
A5191,0005
0.05 mg (= 250 µl)
A5191,0025
0.25 mg (= 1.25 ml)
Assay conditions:
1.7 % agarose
DNA Ladder 100 bp equalized (lyophilised)
A3302
DNA size standard for medium-sized fragments and PCR products
Description
With this marker, the band mass of the larger fragments have been reduced and have
been reinforced for the smaller fragments (as compared to marker A3470) by up to
a factor or 4.5. The result is an even appearance in intensity of the individual bands.
The mol number of the fragments decreases from the larger to the smaller fragments,
as do the possible marking positions on the terminal EcoR I generated recessed ends.
The 500 bp band, with an increase in mass of a ­factor of 3, enables rapid orientation on
1.5-2.0 % agarose gels. Thanks to the even distribution, a shorter migration distance is
needed for the larger fragments, in order to achieve a clear ­distinction between the bands.
The best results are achieved after a migration distance of approx. 60-80 mm on agarose
gels. Each lane of a normal sized ­agarose gel (approx. 80 ml) should be loaded with approx.
0.2-0.5 µg of marker. The ­equalized 100 bp ladder is ­therefore very economical in use.
Loading buffer is supplied separately.
Number of bands
11
Fragment sizes (bp) 1000, 900, 800, 700, 600, 500 (x3), 400, 300, 200, 150, 100
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA;
loading buffer (1X) 10 % glycerol; 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.2-0.5 µg per lane
Storage
-20°C
Package size
A3302,0020
20 µg
DNA marker
6
Size Marker • © 2010 AppliChem
DNA Ladder 100 bp plus
Description
Number of bands
Fragment sizes (bp)
Concentration
Storage
Loading
Package sizes
A5216
100 bp + 1.5 kb DNA Ladder is suitable for sizing linear doubles­ tranded DNA fragments from 0.1 to 1.5 kb. Use in combination with a
loading buffer (please see our present catalog for a list of loading
buffers).
11
1500, 1000 (x2), 900, 800, 700, 600, 500 (x2), 400, 300, 200, 100 (x2).
The 500 bp and 1 kb bands are brighter than the other bands in the ladder.
The marker is supplied in a concentration of 0.2 mg/ml
in 10 mM Tris · HCl (pH 8.0), 1 mM EDTA, 10 mM NaCl.
Store at -20°C. Prepare small aliquots to prevent repeated
freeze & thawing!
We recommend loading of 0.4-0.6 µg (2-3 µl) per lane.
A5216,0005
0.05 mg (= 250 µl)
A5216,0025
0.25 mg (= 1.25 ml)
DNA Ladder Mix 100-5000 (lyophilised)
Assay conditions:
1.7 % agarose
A3660
DNA size standard for the determination of the size of medium-sized fragments and plasmids
Description
The 100 bp ladder (A3470) was extended with fragments in the size range
of 1500-5000. The extension by 40 % mass in the larger fragments means easy
­orientation from 1500 bp upwards on 1.2-1.5 % agarose gels. In addition to small and
large plasmid insertions and PCR products, the size and quantity of plasmid­ ­vectors
can also be determined. The best results are achieved after a migration distance
of 80-90 mm on agarose gels with loading volumes of 0.5-0.8 µg per lane on
standard sized gels.
Number of bands
17
Fragment sizes (bp) 5000, 4000, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600,
500 (x2), 400, 300, 200, 150, 100.
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA;
loading buffer (1X) 10 % glycerol; 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.5-0.8 µg per lane (for good staining properties with ethidium bromide)
Migration distance Best results are achieved after a migration distance of 70-90 mm.
Storage
-20°C. Avoid repeated thawing and refreezing. Portioning in small aliquots is
recommended. The lyophilised product is stable for many weeks even at room
temperature!
Package size
A3660,0050
50 µg
This sizing standard was manufactured using digestion of 7 plasmids with EcoR I. The plasmids are legally
p­ rotected. Permission to produce copies must be sought from the manufacturer. The DNA was deproteinised,
­precipitated and lyophilised after digestion with phenol/chloroform. If the ­fragments are to be labeled, the­ marker
should be dissolved in distilled water (10 minutes at room temperature, shaking occasionally).
© 2010 AppliChem • Size Marker
7
DNA Ladder 250 bp (lyophilised)
A3982
Universal DNA size standard for the determination of the size of plasmids and their DNA insertions
Description
With its two clearly reinforced bands at 2500 and 1500 base pairs, the 250 bp ladder enables
rapid orientation on 1.0-1.2 % agarose gels. Using gel electrophoresis, with intervals of 250 base
pairs in a range of 250-3000 base pairs, it is possible after restriction to determine clearly and
precisely, for example, large plasmid insertions, and vector plasmids above 3000 base pairs with
intervals of 1-2 kb. In comparison to DNA markers with equimolar band distribution, the mass of
the smaller bands of this 250 bp ladder up to 1000 bp has been reinforced and the larger bands
above 3000 bp have been reduced. This means that optimum results are already achieved after
a migration distance of 60-80 mm on agarose gels and that small amounts of about 0.7 µg per
lane can be used with standard sized agarose gels (80 ml).
Number of bands
16
Fragment sizes (bp) 8000, 6000, 5000, 4000, 3000, 2750, 2500 (x3), 2250, 2000, 1750, 1500 (x3), 1250, 1000, 750,
500, 250
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.4-0.8 µg per lane (for good staining properties with ethidium bromide)
Migration distance Best results are achieved after a migration distance of 75-85 mm.
Storage
-20°C. Avoid repeated thawing and refreezing. Portioning in small aliquots is recommended. The
lyophilised product is stable for many weeks even at room temperature!
Package size
A3982,0050
50 µg
This sizing standard was manufactured using digestion of plasmids with EcoR I. The plasmids are legally protected.
Permission to produce copies must be sought from the manufacturer. The DNA was deproteinised, precipitated and lyophilised
after digestion with phenol/chloroform. If the fragments are to be labeled, the marker should be dissolved in distilled water
(10 minutes at room temperature, shaking occasionally).
DNA Ladder 250 bp plus (lyophilised)
A8640
DNA size standard for genomic DNA, plasmids and their insertions
DescriptionThe DNA Ladder 250 bp plus is intended for use in the sizing of DNA fragments during cloning and of genomic DNA. By combining two groups of fragments differing in the
fragment size range, smaller fragments (250-2000 bp) as well as plasmid vectors and
fragment of genomic DNA (3000-12000 bp) my be sized on the same gel. The mass
of the larger fragments is reduced to achieve a better separation on short distances of
70-90 mm only. The terminal Eco RI restriction sites allow for an efficient labeling of
the fragments. Best results are achieved by loading 0.8-1.0 µg/lane on a 1.2 % agarose
gel (standard minigel size). The DNA is supplied deproteinized and lyophilized.
Number of fragments 15 bands
Fragments sizes (bp)12000, 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1750, 1500, 1250, 1000, 750,
500, 250
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.4-0.8 µg/lane (e.g. 1.0-1.2 % agarose gel)
Storage
Store at -20°C. Store as small aliquots, if resuspended.
Package size
A8640,0050 50 µg
8
Size Marker • © 2010 AppliChem
DNA Ladder 1 kb (lyophilised)
A2667
Description The size of medium-sized plasmids and fragments can be determined with 11 DNA bands
between 10,000 and 500 base pairs. Unlike the equimolar distribution of the bands in other
DNA markers and the long migration distance necessary with these for the separation
of large ­fragments, the mass of the larger bands of the 1 kbp DNA ladder has been
reduced. This permits relatively short separation distances for a 1 kbp DNA ladder
(approx. 70 mm on 1.2 % agarose gels) and means that it is very economical in use
(0.4-0.6 µg per lane, or 400 separations) on normal sized gels. Loading buffer is
supplied separately. The fragments have terminal EcoR I generated recessed ends.
The DNA is deproteinized and lyophilized.
Number of bands
11
Fragment sizes (bp): 10000, 8000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 500
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.4-0.6 µg per lane
Storage
-20°C
Package sizes
A2667,0050
50 µg
A2667,0200
4 x 50 µg
DNA marker
DNA Ladder 1 kb
A5207
Description
1 kb DNA Ladder is a convenient marker for determining the size of
d ouble-stranded DNA from 250 to 10,000 base pairs. The 1,000 and
3,000 bp fragments have increased intensity relative to the other bands
on ethidium bromide-stained agarose gels, and serve as reference
­indicators. All fragments are blunt-ended.
Number of bands
13
Fragment sizes (kb) 10.0, 8.0, 6.0, 5.0, 4.0, 3.0 (x2), 2.5, 2.0, 1.5, 1.0 (x2), 0.75,
0.5, 0.25
Storage buffer
10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
Concentration
0.2 mg/ml
Storage
-20°C
Package sizes
A5207,0005
0.05 mg (= 250 µl)
A5207,0025
0.25 mg (= 1.25 ml)
© 2010 AppliChem • Size Marker
9
DNA Ladder 1 kb concatamer (lyophilised)
A6430
DNA size marker as alternative to irregular l-fragments (from 1 to approx. 25 kb)
Description
A partial ligation of 1000 bp Hind III fragments results in a DNA ladder ranging from 1000 to
approx. 22000 bp in 0.8 to 1% gels using standard agarose (Prod.-No. A2114; separation
range up to approx. 25 kbp). The upper limit of the fragment ligation results in the main
mass of the ligation products in the separation range optimal for standard agaroses (agar).
For a better orientation, the 3000 bp and 10,000 bp bands are brighter. Optimal results will be achieved
after a distance of approximately 80-100 mm in agarose gels and a loading volume of approximately
1 µg per lane in normal-sized gels. This 1000 bp concatamer ladder may be applied either in reduced
loading volumes (for a seperation range e.g. < 10000 bp) or in increased volumes (> 15000 bp).
Even under optimized ligation conditions, an intramolecular religation may occur, especially with
smaller fragments. Therefore, at high gel loading volumes, ligation products (closed circles) have been
observed between the 1000 and 3000 bp bands. For a better orientation, the 3000 bp band is brighter.
Number of bands
> 25
Fragment sizes (bp) 1 000 bp steps (1000 - approx. 25000)
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
approx. 1.0 µg per lane
Migration distance Optimum results after a distance of 15-25 mm only.
Storage
-20°C.
Package size
A6430,0050
50 µg
This marker has been designed as an alternative for irregular λDNA fragment markers, for the separation in standard low endoosmosis agarose
(Prod.-No. A2114; separation range 70 bp to approximately 25 kbp). In comparison to other concatamer markers, it has a reduced mass in the
non-separating range (high molecular weight range), resulting in a better resolution (good readings up to 24 kbp). For applications requiring
a good separation in the high molecular weight range pulsed-field electrophoresis is recommended. Due to the production procedure (ligation
of 1000 bp Hind III fragments), it is impossible to determine the specific mass of a single band. The DNA of this marker (50 µg sufficient for
approximately 100 loadings) is deproteinised, Iyophilised and is supplied with 1 ml of the 1 x gel loading buffer.
DNA Marker quick-run (lyophilised)
A7215
DNA size standard for determination of DNA fragments after short gel runs (15 - 25 mm)
Description
This marker is ideal for use with mini gels, since its bands are separated very well even
after very short gel runs as short as 15-25 mm in an e. g. 1.5-1.8 % agarose gel. The
single bands have been equalized in terms of their mass. The 1500 bp fragment has
an increased mass for better orientation. Optimum results are achieved in a 1.5-1.8
% agarose gel with as little as 0.25 µg of the marker per lane (normal sized mini gel).
Please note that the DNA fragments have to be saturated with ethidium bromide in
the running buffer during the separation.
Number of bands
5
Fragment sizes (bp) 2500, 2000, 1500 *, 1000, 500
The mass of the marked fragment* has been increased for better orientation.
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.015 % bromophenol blue
Loading volume
0.25 µg per lane (for a good detection with ethidium bromide)
Migration distance Optimum results after a distance of 15-25 mm only.
Storage
-20°C. Prevent repeated freezing and thawing (> 20x). We recommend to prepare
small aliquots. The lyophilised form is stable even for weeks at ambient temperature
Package size
A7215,0050
50 µg
This marker is made of plasmids with specific mutagenesis sites. The Eco RI-digested DNA has been deproteinized with phenol/chloroform,
desalted, precipitated and lyophilised. Resuspend the DNA in the sterile-filtered gel loading buffer (supplied with the marker) by incubation for
10 minutes at room temperature with occasional shaking.
10 Size Marker • © 2010 AppliChem
DNA Marker quick-run extended (lyophilised)
A7222
DNA size standard for determination of DNA fragments after short gel runs (35 mm)
DescriptionThis marker is ideal for use with mini or midi gels, since its bands are separated
very well even after very short gel runs as short as 35 mm in an e. g. 1.2 % agarose
gel. The single bands have been equalized in terms of their mass. The 1500 bp
fragment has an increased mass for better orientation. Optimum results are achieved
with as little as 0.3-0.5 µg of the marker per lane (normal-sized mini/midi gel).
Number of bands
9
Fragment sizes (bp) 6000, 4000, 2500, 2000, 1500*, 1000, 500, 200, 100
The mass of the marked fragment* has been increased for better orientation.
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.015 % bromophenol blue
Loading volume
0.3 - 0.5 µg per lane
(for a good detection with ethidium bromide in a mini or midi gel)
Storage-20°C. Prevent repeated freezing and thawing (> 20x). We recommend to prepare
small aliquots. The lyophilised form is stable even for weeks at ambient temperature!
Package size
A7222,0050
50 µg
This marker is made of plasmids with specific mutagenesis sites. The Eco RI-digested DNA has been deproteinized with phenol/chloroform, desalted, precipitated and lyophilised. Resuspend the DNA in the sterile-filtered gel
loading buffer (supplied with the marker) by incubation for 10 minutes at room temperature with occasional
shaking.
DNA marker
DNA Marker pBR322 - Hae III (lyophilised)
A4406
DNA size standard for high-percentage agarose gels and polyacrylamide gels
Description
Plasmid pBR322 was digested with Hae III, deproteinized and lyophilized. This
marker is particularly suited to comparisons with small PCR fragments and DNA
from restriction samples on polyacrylamide gels or high-percentage agarose gels
(> 2.2 %). The best results are achieved after a migration distance of approx.
70-80 mm on agarose gels or 100 mm on 6-8 % polyacrylamide gels. Each lane of
a normal sized gel (approx. 80 ml) should be loaded with approx. 1 µg of ­marker
for agarose gels or 0.5 µg for PAA gels. Loading buffer is supplied separately.
Number of bands
22
Fragment sizes (bp) 587, 540, 502, 458, 434, 267, 234, 213, 192, 184, 124, 123, 104, 89, 80, 64, 57,
51, 21, 18, 11, 8
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.5-1.0 µg per lane
Storage
-20°C
Package size
A4406,0050
50 µg
Please note! Extremely small fragments can only be visualized on high-percentage PAA gels.
© 2010 AppliChem • Size Marker
11
DNA Marker pBR322 - Hae III
Description
Number of bands
Fragment sizes (bp)
Storage buffer
Concentration
Storage
Package sizes
A5229
This marker is generated by digestion of the plasmid pBR322 with Hae III.
22
587, 540, 502, 458, 434, 267, 234, 213, 192, 184, 124, 123, 104, 89, 80, 64, 57,
51, 21, 18, 11, 8
10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
0.2 mg/ml
-20°C
A5229,0005
0.05 mg (= 250 µL)
A5229,0025
0.25 mg (= 1.25 ml)
Assay conditions:
1.7 % agarose
DNA Marker pBR328 Mix (lyophilised)
A6927
DNA size standard for middle-sized fragments and PCR products
Description
Plasmid pBR328 was digested with Hinf I and Bgl I, respectively, deproteinised and
lyophilised. The fragments have been mixed in an equimolar ratio. This marker is
particularly suitable for comparison with PCR fragments (e.g. food diagnostics) and
DNA from restriction samples on agarose gels with sizes between 150 and 2000 base
pairs. The concise distribution of fragments of the marker makes this product ideal
for long and short gel runs with running distances of 70-80 mm (1.5 % agarose
gel). Load 1 µg of the marker per lane of a normal sized agarose gel (approx. 80 ml
Loading buffer is supplied separately.
Number of bands
12
Fragment sizes (bp) 2176; 1766; 1230; 1033; 653; 517; 453; 394; 298; 234; 220; 154
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
1.0 µg per lane
Storage
-20°C. Prevent repeated freezing and thawing (> 20x). We recommend to prepare
small aliquots. The lyophilised form is stable even for weeks at ambient temperature!
Package sizes
A6927,0050
50 µg
Resuspend the DNA in the sterile-filtered gel loading buffer (supplied with the marker) for 10 minutes at room
temperature by occasional shaking.
DNA
12 Size Marker • © 2010 AppliChem
DNA Marker Phage Lambda - Sty I
Description Number of bands
Fragment sizes (bp)
Storage buffer
Concentration
Storage
Package sizes
A5194
Lambda DNA (cI857 Sam 7) Sty I markers are prepared by digesting Lambda
DNA with Sty I, followed by inactivation of the enzyme. The DNA fragments
are then ethanol-precipitated and resuspended in the storage buffer.
11
19329*; 7743; 6223; 4254*; 3472; 2690; 1882; 1489; 925; 421; 74
10 mM Tris · HCl (pH 8.0), 1 mM EDTA
0.2-0.5 µg/µl
-20°C
A5194,0005
0.05 mg (= 250 µl)
A5194,0025
0.25 mg (= 1.25 ml)
Please note! The cohesive ends of fragments 1 and 4 (*) may cause the formation of an extra band (23583 bp).
The fragments may be separated by heating to 65°C for 3 minutes before loading the sample onto the gel.
DNA Marker Phage Lambda - BstE II (lyophilised)
A4412
DNA size standard made of phage λ DNA for the determination of large DNA fragments (digestion of genomic DNA)
Description Natural lambda DNA was digested with BstE II, deproteinized and lyophilized. This
arker is suitable for comparisons with fragment sizes between 8400 and 700 base
m
pairs. The best results are achieved after a migration distance of approx. 80-90 mm
on 1.0-1.2 % agarose gels. Each lane of a normal sized gel (approx. 80 ml) should
be loaded with approx. 1 µg of marker. Loading buffer is supplied separately.
Number of bands
14
Fragment sizes (bp) 8454*, 7242, 6369, 5686*, 4822, 4324, 3675, 2323, 1929, 1371, 1264, 702,
224, 117
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
1.0 µg per lane
Storage
-20°C
Package size
A4412,0100
2 x 50 µg
Please note! The cohesive ends of fragments 1 and 4 (*) may form an additional band (14140 bp). Before loading
onto the gel, the fragments can be separated by heating to 65°C for 5 minutes with subsequent incubation on ice.
marker
© 2010 AppliChem • Size Marker
13
DNA Marker Phage Lambda - BstE II
Description Number of fragments
Fragment sizes (bp)
Storage buffer
Concentration
Storage
Package sizes
A5220
Phage Lambda (cI857 Sam 7)DNA/BstE II markers are prepared by digesting
L ambda DNA with BstE II, followed by inactivation of the enzyme. The DNA
­fragments are then ethanol-precipitated and resuspended in the storage buffer.
14
8454*, 7242, 6369, 5686*, 4822, 4324, 3675, 2323, 1929, 1371, 1264, 702,
224, 117
10 mM Tris · HCl (pH 8.0), 1 mM EDTA
0.2-0.5 µg/µl
Store at -20°C
A5220,0005
0.05 mg (= 250 µl)
A5220,0025
0.25 mg (= 1.25 ml)
Please note! The cohesive ends of fragments 1 and 4 (*) may cause the formation of an extra band (14140 bp).
The fragments may be separated by heating to 65°C for 3 minutes before loading the sample onto the gel.
DNA Marker Phage Lambda - Hind III (lyophilised)
A5589
DNA size standard made of phage λ DNA for the determination of large DNA fragments (digestion of genomic DNA)
Description Natural lambda DNA was digested with Hind III, deproteinized and lyophilized. This
marker is suitable for comparisons with large to medium-sized fragments between
23,000 and 600 base pairs. The best results are achieved after a migration distance of
approx. 80-90 mm on 1.0-1.2% agarose gels. Each lane of a normal sized gel (approx.
80 ml) should be loaded with approx. 0.5-0.8 µg of marker. Loading buffer is
supplied separately.
Number of bands
8
Fragment sizes (bp) 23130*; 9416; 6557; 4361*; 2322; 2027; 564; 125
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.5-0.8 µg per lane
Storage
-20°C
Package size
A5589,0100
2 x 50 µg
Please note! The cohesive ends of fragments 1 and 4 (*) may form an additional band (27491 bp). Before loading
onto the gel, the fragments can be separated by heating to 65°C for 5 minutes with subsequent incubation on ice.
DNA marker
14 Size Marker • © 2010 AppliChem
DNA Marker Phage Lambda - Hind III
A5223
Description L ambda DNA (cI857 Sam 7)/Hind III markers are prepared by digesting Lambda DNA with
Hind III, followed by heat-inactivation of the enzyme. The DNA fragments are then ethanolprecipitated and resuspended instorage buffer.
Number of bands
8
Fragment sizes (bp) 23130*; 9400; 6557; 4361*; 2322; 2027; 564; 125
Storage buffer
10 mM Tris · HCl (pH 8.0), 1 mM EDTA
Concentration
0.2-0.5 µg/µl
Storage
-20°C
Package sizes
A5223,0005
0.05 mg (= 250 µl)
A5223,0025
0.25 mg (= 1.25 ml)
Please note! The cohesive ends of fragments 1 and 4 (*) may cause formation of an extra band (27491 bp).
The fragments may be separated by heating to 65°C for 3 minutes before loading the sample onto the gel.
DNA Marker pUC19 - Msp I
Description
Number of bands
Fragment sizes (bp)
Storage buffer
Concentration
Storage
Package sizes
A5235
Digestion of the pUC19 plasmid yields 12 fragments.
12 (9 visible)
501, 489, 404, 331, 242, 190, 147, 111, 110, 67, 34x2, 26
10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
0.2 mg/ml
-20°C
A5235,0005
0.05 mg (= 250 µl)
A5235,0025
0.25 mg (= 1.25 ml)
Assay conditions:
1.7 % agarose
DNA Marker pUC19 - Msp I (lyophilised)
A3996
DNA size standard for high-percentage agarose gels and polyacrylamide gels
Description Plasmid pUC19 was digested with Msp I, deproteinized and lyophilized. This marker is particularly suitable for comparisons with small PCR fragments and DNA from restriction samples on
polyacrylamide gels or high-percentage agarose gels. The bands at 501/489 bp and 111/110 bp
have approx. double mass and provide rapid orientation after a short migration distance on 2.02.2 % agarose gels. The best results are achieved after a migration distance of approx. 50-70 mm
on agarose gels or 100 mm on 6-8 % polyacrylamide gels. Each lane of a normal sized agarose
gel (approx. 80 ml) should be loaded with approx. 0.5-1.0 µg of marker. Loading buffer is
supplied separately.
Number of bands
12
Fragment sizes (bp) 501, 489, 404, 331, 242, 190, 147, 111, 110, 67, 34, 26
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume
0.5-1.0 µg per lane
Storage
-20°C
Package size
A3996,0050
50 µg
Please note! Extremely small fragments can only be visualized using high-percentage and high-quality agarose gels (> 2.2
%) with large mass of fragments.
© 2010 AppliChem • Size Marker
15
2.1 dyes for nucleic
Prod.-No.
Description
DNA/RNA Dye Complex
A1398
Acridineorange
dsDNA/RNAgreenfluorescence;
ssDNA/RNAredfluorescence
A0691
Crystalviolet
DNA(purple-red)
A1001
DAPI
specificbindingtoAT-Basepairs;
intercalationintoGC-Basepairs;white-bluefluorescence
A1151
Ethidiumbromide*
DNAintercalation
A2388
Malachitegreenoxalate
DNA
A1402
Methyleneblue
DNA,RNA-stainingatacidicpH
A5595
DNA-DyeMethyleneblue
DNA
A1403
Methylgreen
specificbindingtoAT-richsequences;DNA(green)
A0581
Methylorange(C.I.13025)
A3918
Nileblue
DNAintercalation(blue)
A2261
Propidiumiodide
DNA-intercalator;nouptakeintolivingcells
A1406
PyroninY
DNA/RNA(red)
A3944
Silvernitrate
DNA(brown)
A1400
Stainsall
RNA(blue-violet)
*Thereareseveralready-to-usesolutionsavailable!
A1152Ethidiumbromide-Solution1%BioChemica
A2273Ethidiumbromide-Solution0.07%“dropper-bottle”
*
Excitation/Emission (Detection)
490nm/525nm
590nm
365nm/450nm
260-360nmor546nm/590nm
626nm
297nm/672nm
297nm/672nm
638nm
630nm/673nm
530nm/625nm
488-530nm/565-574nm
DNA-Dye Methylene blue
A5595
Nontoxicsolutionforthestainingofnucleicacidsinagarosegels
Amount
10ml200-foldmethylenebluedyeconcentrate
Storage
2-8°C
Stability
18-24months(shakewellbeforeuse)
Package size
A5595,0010
10ml
Protocol for staining with DNA-Dye Methylene blue:
The DNA marker
Phage Lambda-Hind III
(A5589) was loaded
on the 1st lane (*).
16
Approx.50ml1XDNA-DyeMethylenebluestainingsolutionarerequiredtostainanagarose
gelwiththedimensionsofapprox.80x60mm(widthxlength)andathicknessof3-4mm.
The gel should be trimmed down appropriately, with the edge of a ruler for example, to
removeareasofthegelthatwerenotusedforDNAseparation.Thestainingisperformedinan
adequatelysizeddish,inwhichthegelisfloatedtoadepthofabout5mmwithdyesolution.
To prepare the DNA-Dye Methylene blue staining solution for use, 250 µl are dissolved in
50mlwater.Thegelislefttostainforabout20minutesandthedishisshakenoccasionally.
Torevealthebands(differentstainingintensities)thegelisdestainedwithtapwaterseveral
timesforabout5-10minutes.Theblue-stainedDNAbandsareclearlyvisibleintransmittedlightandcanbe
measuredwitharulerordocumentedphotographically.
MethylenebluedoesnotstainDNApermanently:dependingonthethicknessofthegelandthestoragetemperature,theDNAbands(especiallysmallerfragments)maylosetheirstainingaftersometime.
SizeMarker•©2010AppliChem
2.2
acids
Sensitivity
50ng
recommended working concentration
30µg/ml
10ng
70ng
1µg/ml
0.1µg/mlinwater
0.5ng
40ng
40-80ng
10ng
40ngonagarose;4ngondriedgel
2.5ngdsDNAonagarose
0.2-0.5µg/ml
0.2%in0.4MNaOAc/0.4MAceticacid
1µg/ml
0.0005%
1µg/ml
1µg/ml
0.05%or1µg/ml
Stock solution / Storage
1µg/mlinwater;2-8°C
10mg/mlinwater;2-8°C
200-foldconcentratedsolution;2-8°C
0.25%inwater
1µg/mlin0.25XTAEbuffer(pH8.0)
2-8°CorRT
Abbreviations
DAPI(4',6-Diamidino-2-phenylindoledihydrochloride);PBS(Phosphate-bufferedsaline);NaOAc(Sodiumacetate)
PBS(Phosphate-bufferedsaline)
Repeated use of the 1X staining solution
The prepared 1X methylene blue staining solution can be kept in a suitable brown-glass bottle at 2-8°C for
4-6monthsandcanbeusedseveraltimes.Thestainingperiodshouldbelengthenedbyabout20%fortheseconduse,and
bythesameagainforthethirduse.Afterthis,thestainingcapacityofthesolutionislargelyexhaustedandanewsolution
shouldbeprepared.
Sensitivity
Thebandsina1kbladder(loadingamount1µg)canbestainedwithnoproblems.Smallfragments(150bp)canalsobe
stained.Thedecisivefactorsinsensitivityarethethicknessofthegel,thegel:stainingsolutionratio,andthedestainingtime.
Byvaryingthestainingconditions,youcaneasilydeterminethebestconditionsforyoursystem.
Handling
Alaboratorycoatandglovesshouldbewornwhenhandlingthestainingsolution.
Precaution
Oncontactwiththeskinorclothing,theareashouldbewashedwithlargeamountsofsoapandwater.
Short protocol
1. DiluteDNA-DyeMethyleneblue200Xconcentratedownto1X(250µlDNA-DyeMethyleneblue+50mlwater)
2.Trimdowntheagarosegeltothesmallestareapossible.
3.Incubate the gel (dimensions approx. 80 x 60 mm) with 50 ml DNA-Dye Methylene blue for about 20 minutes;
shakeoccasionally.Thestainingsolutionshouldcoverthegeltoadepthofabout5mm.
Please note:thestainingsolutioncanbeusedseveraltimes!
4.Destainrepeatedlyinwaterfor5-10minutes.
©2010AppliChem•SizeMarker
17
dyes for nucleic acids
2.3
18
Ethidium bromide
isthemostcommonlyuseddyeforstainingDNAduringorafterpolycrylamideoragarosegelelectrophoresis.
Excitationwithlightatawavelenghtof254nmisabsorbedbytheDNAandtransferredtoethidiumbromide.
Excitationat366nmdirectlyexcitesthedye(3).Anaqueousstocksolutionispreparedataconcentrationof
10mg/mlandemployedat0.2-0.5µg/ml(5).Stainingisperformedafterthegelrun(especiallyPAGE)or
duringthegelrun(agarose).ThelatterallowstheobservationofthegelrunbycontrolunderUVlight.Ethidium
bromideisaddedtotheagarosesolution.Pleasenote,that'prestaining'mayleadtoartifacts(4).
In another application, ethidium bromide may be used to sensitively quantify DNA by spectrofluorimetry.
Excitationat250nmwithUVlightandanemissionat605nm,thesensitivityisevenbetterascomparedtothe
dyeHoechst33258.Ataconcentrationof0.5µg/mlEtBr,thedetectionlimitof10ng/mlDNAisreachedwitha
linearrangeofmeasurementfrom20to1250ng/ml(6).
Caution:Ethidiumbromideisapowerfulmutagenandismoderatelytoxic.Glovesshouldbewornwhenworking
withsolutionsthatcontainthisdye,andamaskshouldbewornwhenweighingethidiumbromide.
Ethidium bromide - Solution 1 % BioChemica
Synonym
Composition Storage
Package sizes
Ethidium bromide - Solution 0.07 % “dropper-bottle”
Synonym
Composition
Storage
Description Package sizes
A1152
2,7-Diamino-10-ethyl-9-phenylphenanthridiumbromide-Solution
Ethidiumbromide:10mg/ml
filteredsolution
Storethestocksolutionat2-8°Cprotectedfromlight.
A1152,0025 25ml
A1152,0100 100ml
A2273
2,7-Diamino-10-ethyl-9-phenylphenanthridiumbromide-Solution
Ethidiumbromide:0.7mg/ml
filteredsolution
Storethestocksolutionat2-8°Cprotectedfromlight.
EthidiumbromideactsasaDNAintercalatorandhasaconsiderablemutagenic
potential.InordertoreducethepossiblecontacttoethidiumbromideAppliChem
providesaready-to-usesolutionina'dropperbottle'.Theconcentrationofethidium
bromidesolution(0.7mg/ml)isadjustedsothattheadditionofonedrop(ofa
volumeof50µl)ofthissolutionissufficienttostain50mlagarosegelsolution.
A2273,0005
5ml
A2273,0015
15ml
Literature
(1) Waring, M. (1975) in Antibiotics Vol. III , 141-165 (J.W. Corcoran & F.E. Hahn eds.) Springer-Verlag: Review article about ethidium and propidium.
(2) Lunn, G. & Sansone, E.B. (1987) Anal. Biochem. 162, 453-458 Ethidium bromide: Destruction and decontamination of solutions.
(3) Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring
Harbor, New York.
(4) Gärtner, U. et al. (1996) Anal. Biochem. 243, 194-196 Apparent degradation of cleaved genomic DNA may be an ethidium bromide prestaining artifact.
(5) Lucey, M.J. et al. (1997) BioTechniques 23, 780-782 Reducing the ethidium bromide quantity in agarose gel electrophoresis.
(6) Bonasera, V. et al. (2007) BioTechniques 43, 173-176 Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using EtBr.
SizeMarker•©2010AppliChem
protein marker
General Considerations
Sizeestimation:Prestainedproteinmarkersarenotrecommendedforprecisedeterminationofthemolecular
weightsizesincetheirbehaviorduringelectrophoresisstronglydependsonelectrophoresisconditions.For
exactdeterminationsofproteinsizesuseunlabeledmarkerproteins,i.e.non-prestainedmarkers.
Proteinblotting:Thetransferofproteinsduringblottingdependsontheirsize(e.g.lysozymeisfullytransferredafter30minutes,whilemyosinrequires2.5hours@1V/cm2).
Prod.
No.
A5238
A5418
A4402
A3993
A8359
A8889
Protein Marker
Bands
ProteinMarkerI(14-116)
ProteinMarkerII(6.5-200)prestained
ProteinMarkerIII(6.5-200)
ProteinMarkerIV(10-150)
ProteinMarkerV(10-175)prestained
ProteinMarkerVI(10-245)prestained
7
8
8
8
11
12
Fragment Sizes [kDa]
3.1
116 97.4 66.2 37.6 28.5 18.4 14
200 116 68 43 30 20 14.4 6.5
200 116 68 43 30 20 14.4 6.5
150 100 80 60 40 30 20 10
175 130 95 70 62 51 42 29 22 1410.5
245 180 135 100 75 63 48 35 25 20 17 11
3.2 precision
Protein Marker I (14-116)
A5238
Description
Thebandsof3µlmarkerareeasily
visualizedwithCoomassiestainingina
polyacrylamidegel.ProteinMarkerIisa
mixtureof7purifiedproteinssuppliedingel
loadingbufferfordirectapplicationto
anSDSpolyacrylamidegel.
Number of bands
7
Fragment sizes (kDa) 116.0;97.4;66.2;37.6;28.5;18.4;14.0
Loading buffer
50mMTris·HCl(pH6.8),
100mMdithiothreitol,2%SDS,
0.1%bromophenolblue,10%glycerol
Assay conditions
3µl/12%PAGE
Package size
A5235,0500 500µl
©2010AppliChem•SizeMarker
19
Protein Marker III (6.5-200)
Description
Number of bands
Protein sizes (kDa)
Assay conditions
Storage
Package size
A4402
Thebandsofthe2.5µlmarkerareeasily
visualizedwithCoomassiestainingina
polyacrylamidegel.
8
200.0;116.0;68.0;43.0;30.0;20.0;
14.4;6.5
5µl/4-20%SDS-PAGEgradientgel;
Coomassiestaining
-20°C,ifstoredlongerthan1month.
Pleasenotethatrepeatedfreezingandthawingwillreduceproductquality.Preparationofsmallaliquotsisrecommended.
A4402,0001 1.0ml
Thisisaready-to-usemarkercontainingacylatedproteins.
Theproductmaycontainresidualiodoacetamide.
precision
Protein Marker IV (10-150)
Application
●Theproteinemarkers
havetobe“preheated”
toroomtemperature,
toguaranteethatallcomponentsaredissolved.
●Apply1-5µlofthe
markertoamini-gel
(10x10cm,1-0.75mm
thick,7mmslot).
Use1XLaemmlibuffer
todilutethismarker,
ifnecessary.
●Tominimizemyosin
aggregation,thealiquotto
beloadedontothegel
maybeheatedto95°C
for1-2minutes.
A3993
Recombinantproteinsizemarkerforgelelectrophoresis
Description
The bands of 2.5 µl marker are easily
visualized with Coomassie staining in a
polyacrylamidegel.
Number of bands
8
Protein sizes (kDa) 150;100;80;60;40;30;20;10
Assay conditions
10µl/4-20%SDS-PAGEgradientgel
(Tris-Glycine);Coomassiestaining
Storage
-20°C, if stored longer than 1 month.
Pleasenotethatrepeatedfreezingand
thawingwillreduceproductquality.
Preparationofsmallaliquotsis
recommended.Thawedaliquotsarestable
foroneweekat+4°C.
Package size
A3993,0500 500µl
Thisisa ready-to-usemarkercontainingrecombinantproteins.
Theproductmaycontainresidualiodoacetamide.
20
SizeMarker•©2010AppliChem
prestained
Protein Marker II (6.5-200) prestained
A5418
Prestainedproteinsizemarkerforgelelectrophoresis
Description
This is a ready-to-use marker containing
covalent prestained proteins. The product
containsformamide.
Number of bands
8
Protein sizes (kDa) 200.0;116.0;68.0;43.0;30.0;20.0;
14.4;6.5
Assay conditions
10µl/4-20%PAGEgradientgel
Tris-Glycine;leftlaneprestainedbutnot
Coomassie-stained;rightlaneprestained
andadditionalCoomassiestaining.
Storage
-20°C,ifstoredlongerthan1month.
Pleasenotethatrepeatedfreezingand
thawingwillreduceproductquality.Pre
parationofsmallaliquotsisrecommended.
Package size
A5418,0250 250µl
3.3
Protein Marker V (10-175) prestained
A8359
PrestainedproteinmarkerforgelelectrophoresisandWestern
blotting.MagentaMarker
Description
Ready-to-useprestainedproteinsizemarker.Suppliedinloadingbuffer.
Number of bands
11
Protein sizes (kDa) 175;130;95;70;62;51;42;29;22;14;10.5
Threereferenceproteinscoupledwithabluedyeforeasyidentification:
approx.10,40,and90kDa.
Storage
At2-8°Cformax.3months;at-20°Cforlongtermstorage.
Package size
A8539,0250 250µl
©2010AppliChem•SizeMarker
21
Protein Marker VI (10-245) prestained
A8889
PrestainedproteinmarkerforgelelectrophoresisandWesternblotting.Blue-Green-RedProteinMarker
Description
ProteinMarkerVIprestainedisathree-colorproteinstandardwith12prestained
proteinscoveringawidemolecularweightrangefromapprox.10to245kDa.
Proteinsarecovalentlycoupledwitheitherabluechromophoreoronegreen
(25kDa)andonereddye(75kDa),respectively.Gelrunmaybemonitored
duringproteinseparationonSDS-PAGE(Tris-glycinebuffer,“Laemmlisystem”).
The main applications are the monitoring of protein migration during
SDS-polyacrylamide gel electrophoresis, the verification of Western transfer
efficiency on membranes (PVDF, nylon, or nitrocellulose), and the sizing of
proteins on SDS-polyacrylamide gels and Western blots. The size marker is
suppliedready-to-useingelloadingbuffer.
Number of bands
12
Protein sizes (kDa) 245;180;135;100;75;63;48;35;25;20;17;11
Package size
A8889,0500
500µl
RecommendationsforLoading
1.Thawtheladdereitheratroomtemperatureorat37-40°Cforafewminutestodissolveprecipitatedsolids.Donotboil!
2.Mixthoroughlytoensurethesolutionishomogeneous.
3.LoadthefollowingvolumesoftheladderonSDS-polyacrylamidegel:5µlperwellformini-gels,
2.5µlperwellforblots;10µlperwellforlargegels,5µlperwellforblots.
3.3 prestained
22
SizeMarker•©2010AppliChem
Sometimes,
less is more!
For The Sake Of
The Environment
Manyreagentspreparedinbiomedicalresearch
labsareidealnutrientbrothsforunwanted
germs(bacteria,fungi).Topreventtheirgrowth,
reagentsareeitherautoclaved,sterilefiltered,or
antibiotics/antimycoticsortoxicsubstancesare
added.OneoftheseadditivesisThimerosal,a
mercury-containingmoleculewhichisdangerous
fortheenvironment.Ouroriginalimmunoasssay
buffercontainedthischemicaltoo,butnowwe
havereplaceditbythenontoxicProClin® 300.
Forthesakeoftheenvironment.
For Better Results
Changingthecompositionhasnonegative
influenceontheperformanceoftheproducts.
WithCrossDownandallotherimmunoassay
buffersyouareeveninabettermoodandyour
immunoassaysshowabetterquality.
For Your Safety
Wewouldliketokeepyouhealthysothatyou
stayourcustomer.FYI:Thimerosalisclassified
astoxic.Thelethaldose(rat,s.c.)is9mg/kg,
comparedtoethidiumbromidewithalethaldose
(mouse,s.c.)of110mg/kg.Insomecountries,
Thimerosalisaforbiddenadditive.
©2010AppliChem•SizeMarker
23
Prod.-No. Description
A2176
BismarckbrownR
Absorption maxima
lmax.468nm
A3480
Comment
increasessensitivityofCoomassie®
BrilliantBlueR-250staining
Coomassie®BrilliantBlueG-250 stainsampholytesinIEFgelstoo!*
A1092
Coomassie®BrilliantBlueR-250 stainsampholytesinIEFgelstoo!*
lmax.withprotein549nm,
w/oprotein555nm
A0822
EosinY
A1346
EriochromeblackT
A1401
FastGreenFCF
dyecomplexinacidicpHrange
A2385
A2388
Kongored
Malachitegreenoxalate
stainingintheacidicpHrange
Phosphatasestainingingels
A1405
Nilered
PonceauS
acidicdiazodye
A7808
A3930
Proteo-DyeRuBPS
RhodamineB$
A3972
A1400
Silvernitrate
Stainsall**
brown
stainsdifferenttypesofproteins
indifferentcolors
Zinc-Imidazole
reversiblestaining
dyes for protein gels
4.1
reversiblestaining
560nm
lmax.(pH8.3)615;(MeOH)620nm
lmax.(withprotein)635nm
lmax.(H2O)517-823nm
lmax.617nm
560nm
lmax.forBSA515nm
Abbreviations:BSA(BovineSerumAlbumin);CBB(Coomassie®Brilliantblue);MeOH(Methanol);
RuBPS(Ruthenium(II)tris(bathophenanthrolinedisulfonate));TCA(Trichloroaceticacid);&onlyincombinationwithCoomassie®!
$detectionofphosphoproteins;formationofinsolublephosphatecomplexes(sensitivity:0.1nM)
4.2
Coomassie® Brilliant blue R-250 (C.I. 42660)
Synonym
BrilliantblueR,Xylenebrilliantcyanine
Order No.
Quantity
A1092,0010 10g
A1092,0025 25g
A1092,0100 100g
®registeredtrademarkofImperialIndustriesPLC
Formula
C45H44N3NaO7S2
A1092
M
CAS-No.
825.98g/mol 6104-59-2
Coomassie®BrilliantBlueR-250isoneofthemostcommonlyusedstainsforproteins,aftertheirseparationbypolyacrylamidegelelectrophoresis. The protein-dye complex has an absorption maximum at 549 nm, the dye without protein at 555 nm (in 0.01 M citrate buffer,
pH3).Theintensityinstainingofproteinsprobablydependsonthebasicityofaprotein.Perpositivelychargedaminoacidapproximately
1.5-3moleculesofCoomassie®willbebound.Thisvariationcomplicatetheexactproteindeterminationwithalbuminasastandard,sincethis
proteincontainsmorebasicaminoacidsthanmanyotherproteins.
TheredoexistmanyprotocolsforsensitivestainingprocedureswithCoomassie®(e.g.ref.3,4).Thesensitivityreachesalimitat25ngprotein.
Werecommendthefollowingprotocol:
I. Staining solution: 0.1 % Coomassie® Brilliant Blue R-250 (Prod.-No. A1092), 20 % methanol (or ethanol), 10 % acetic acid.
TheSDSgel(without'stackinggel')isstainedfor1hourat60°Corfor2hoursat50°CorovernightatRT.
II. Destainingsolution:20%methanol(orethanol)and10%aceticacid.Destainthegelfor3-4hoursat50-60°C.Addsomesponges.
Subsequentlywashthegelfor15minutsinwateranddryundervacuumat60°Cfor2-3hours.
24
SizeMarker•©2010AppliChem
Sensitivity
25 ng &
Stock solution
Solvent MeOH : Acetic acid : Water (40:7:53);
dissolve dyes separately, then mix 1:0.75 (v/v)
0.1 % w/v CBB in 2 % w/v Phosphoric aicd,
10 % w/v Ammonium sulfate (Do not filter!)
10 ng
Recommended Concentration
Coomassie Brilliant blue R-250 (0.2 % w/v)
Bismarck brown R (0.05 % w/v)
Mix 80 ml of the stock solution
with 20 ml of MeOH just before use.
0.1 % in 20 % MeOH, 10 % Acetic acid
similar to CBB R-250
0.25 % (w/v) in 0.1 N NaOH
10 ng
0.01 %
prepare staining solution fresh daily;
may be used for several gels;
0.02 % in 40 % MeOH / 7 % Acetic acid;
in combination with Rhodamine B
400 ng
0.25 % (w/v) in 10 % Acetic acid up to 1 %
(w/v) in 7 % Acetic acid
0.1 % (w/v) in 0.2 M Acetate buffer (pH 3.5)
Staining solution =
3 Vol. Solution 1 + 1 Vol. Solution 2
(stable for 3 weeks; filter prior to each use)
< 0.5 ng
less sensitive than CBB
5 ng
500 ng
1 % (w/v) in distilled water
Solution 1: 0.15 g Malachite green oxalate in 300 ml Water;
Solution 2: 4.2 g Ammonium molybdate tetrahydrate in 100 ml
5 N HCl;
prepare fresh prior to use
2-5 ng
10 ng
0.1 - 0.5 % in 3 % TCA or 1 ml Acetic acid
glacial ad 100 ml Water
1 µM
0.01 %
2-5 ng
like CBB
0.005 %
0.1 % in Formamide or 5.6 mg/10 ml in 50 % Dioxane;
prepare fresh
1 mM
0.02 % in 40 % MeOH / 7 % Acetic acid; in combination with
Eriochrome black T
10-20 ng SDS-PAGE
40-80 ng native PAGE
* Allen, R.E. et al. (1980) Anal. Biochem. 104, 494-498. Staining Proteins in Isolelectric Focusing Gels with Fast Green.
** stains e.g. sialoglycoproteins and phosphoproteins blue, almost all other proteins red.
Coomassie® Brilliant blue G-250 (C.I. 42655)
A3480
Synonym Brilliant blue G
Order No. Quantity
Solubility (20°C) Formula
A3480,0010 10 g
~10 g/L (H2O) C47H48N3NaO7S2
A3480,0025 25 g
A3480,0100 100 g
® registered trademark of Imperial Industries PLC
M
CAS-No.
854.04 g/mol 6104-58-1
The assays of Neuhoff et al. (Neuhoff, V. et al. (1988) Electrophoresis 9, 255-262) have shown that stainng with
Coomassie® G-250 is more sensitive than with R-250. For more informations see A1092.
© 2010 AppliChem • Size Marker
25
Proteo-Dye Blue-Vis
Synomym
Storage
HS-No.
Package size
A6810
Proteindyeinthevisiblerange
2-8°Cprotectedfromlight
38220000
A6810,1000 1L
1Lissufficientforapprox.30minigels,since3xreusable
detectionlimit3-5ng/mm²
reversiblestainingofproteins
Protocol for staining of gels with Proteo-Dye Blue-Vis:
SDS-PAGE minigels (1 mm thickness, 10 % acrylamide, Tris-glycine buffer system) are
treatedfor1hourwith30%v/vmethanolafterelectrophoresistoremovetheexcessof
SDSandtofixtheproteinsinthegelmatrix.Thegelsarestainedfor2hoursinavolume
of100mlProteo-DyeBlue-Vis.Afterthis,thegelsarewashedwithacetatebuffer(0.2M;
pH4.5),containing20%v/vmethanolfor90-120minutesuntilthedarkredbackground
isreducedtoaweakpinkbackgroundincontrasttotheblueproteinbands.Thegelsare
documentedwiththehelpofavideosystem(transilluminator312nm,whitelightplate)or
onascannerinthetransmissionmode.
4.3 dyes
Proteo-Dye Red-Fluo
Synomym
Storage
HS-No.
Specification
Package size
A6803
Proteindyewithredfluorescence
2-8°Cprotectedfromlight
38220000
Emissionmaximum:630nm
Exicitationwavelenght:312nm
A6803,1000 1L
1Lissufficientforapprox.30minigels,since3xreusable
detectionlimit1-3ng/mm²
reversiblestainingofproteins
moresensitivethanmostotherproductsavailableinthemarket
Protocol for staining of gels with Proteo-Dye Red-Fluo:
SDS-PAGEminigels(5x8cm,1mmthickness,10%acrylamide,Tris-glycinebuffersystem)
arefixedfor30minutesinasolutionof7.5%v/vaceticacid,20%v/vethanol.Afterthis,
thegelsarestainedwithProteo-DyeRed-Fluo(100ml)for2-3hours.Thebackground
fluorescence can be removed by repeated washing with the fixation solution (7.5 % v/v
aceticacid,20%v/vethanol;3-4washingsteps,30minuteseach).Thedocumentation
of the gels is carried out with a video system (transilluminator 312 nm, adapting filter
590nm).
26
SizeMarker•©2010AppliChem
Proteo-Dye Green-Fluo
Synomym
Storage
HS-No.
Specification
Package size
A6794
Protein dye with green fluorescence
2-8°C protected from light
38220000
Emission maximum: 520 nm
Excitation wave lenght: 365 nm
A6794,1000 1L
1 L is sufficient for approx. 30 minigels, since 3x reusable
detection limit 3-5 ng/mm²
reversible staining of proteins
suitable for gels and for blots
Proteo-Dye Green-Fluo is a fluorescence protein dye, based on a metal-chelate complex in
conjunction with a detergent in submicellar concentration in an aqueous solution. This dye
solution enables to detect very small amounts of proteins and the staining is fully reversible.
Use the dye solution up to three times!
Protocol for staining of gels with Proteo-Dye Green-Fluo:
SDS-PAGE minigels (5x8 cm; 1 mm thickness, 10% acrylamide, Tris-glycine buffer system)
are postelectrophoretically treated for 30 minutes with a methanol solution (30 %, v/v)
while gently shaking (100 rpm). After this, the gels are stained for 2 hours in a volume of
100 ml Proteo-Dye Green-Fluo. Reduction of the background fluorescence is achieved by
repeated washing steps (3-4 times, 30 min. each) with 30 % v/v methanol. The gels are
documented with a video system (transilluminator 365 nm, adapting filter 520 nm).
Protocol for staining of blots with Proteo-Dye Green-Fluo:
By staining with Coomassie or silver the proteins in the gel become irreversibly de­naturated
and are difficult to transfer to blotting membranes. Metal chelate staining methods are
reversible, resulting in good preconditions for the following blot transfer. Blots were
performed by a typical semi-dry method. After the blotting process, the blots were washed
in distilled water for 1 hour and dried at room temperature. Blots were then stained with
Proteo-Dye Green-Fluo for 2 hours, briefly washed in 30 % v/v ethanol and dried again.
Only in the dried state, intensely gree n fluorescent protein bands become visible in UV
toplight (365 nm UV light) and are documented with a video system (adapting filter 420
nm). The stain may be easily and fastly removed just by rinsing in 30% v/v ethanol.
for protein gels
© 2010 AppliChem • Size Marker
27
Proteo-Dye RuBPS
A7808
Synomym
Ruthenium(II) tris(bathophenanthroline disulfonate) tetrasodium salt
Formula
C72H42N6Na4O18RuS6
Molecular weight
1664.58 g/mol
CAS-No.
[301206-84-8]
Concentration (in H2O)
1 mM
lmax. Emission (in H2O)
617 nm
lmax. Exitation
Storage
recommended working solution
Stability
Package size
277, 437, 460 nm
2-8°C, protected from light
1 : 1000 dilution (1 µM final concenrtation)
2 years
A7808,0001
1 ml
Comment
2-D gels of E. coli proteins stained with (1) SYPRO
Ruby© and (2) with RuBPS. For details see Reference 2.
RuBPS is a fluorescence dye for protein detection in e.g. SDS- and 2-D-gels. It provides the high
sensitivity of silver staining without its drawbacks. The excellent contrast, good linearity and
homogeneity made this dye the staining reagent of choice in proteomics. A minimal interference
with MALDI-TOF analysis is observed and compatibility with MS/MS analysis is given. The photochemically stable dye is excited with UV-light of the wavelength 473 or 488 nm blue laser. A 532
nm laser may be used as well, albeit with reduced ­efficiency. It is of advantage that the excitation
wavelength of RuBPS is longer than tose of the aromatic amino acids.
The information given in terms of formula, molecular weight and CAS number refer to the
raw material! The concentrate supplied is diluted 1 : 1000 (1 ml ad 1 L) resulting in an
1 µM working solution.
Special features of Proteo-Dye RuBPS
•
•
•
•
•
•
•
Protocol for gel staining / destaining
Literature
[1] A comparison between Sypro Ruby© and
ruthenium(II) tris(bathophenanthroline disulfonate)
as fluorescent stains for protein detection in gels.
(Rabilloud, T. et al. (2001) Proteomics 1, 699-704)
[2] Improved ruthenium(II)
tris(bathophenanthroline disulfonate) ­staining
and destaining protocol for a better signal-tobackground ratio and improved resolution.
(Lamanda, A. et al. (2004) Proteomics 4, 599-608)
28 Provides the sensitivity of silver-staining without its drawbacks
(limited dynamic range, inhomogeneity and interference with
MS analysis)
Carefully purified product offers much improved signal-tobackground ratio
Excellent contrast in gel images enables protein spots to be
detected more easily by image processing software
High sensitivity
Good linearity and homogeneity
Minimal interference with MALDI-TOF analysis
Compatible with MS/MS analysis, provided that cleaning of the
sample with reverse phase adsorption is carried out
(according to Ref. 2)
1. Prepare a 1 µM Proteo-Dye RuBPS solution by diluting the concentrate 1000-fold
(e.g., 1 ml to 1 L)
2. Fix the gel in 30 % ethanol, 10 % v/v acetic acid overnight.
3. Rinse the gel in 20 % ethanol v/v for 30 min and repeat 3 times.
4. Incubate the gel in 1 µM Proteo-Dye RuBPS solution for 6 h.
5. Equilibrate the gel in water for 10 min and repeat once (a first scan is possible at this
stage).
6. Destain the gel with 40 % ethanol / 10 % acetic acid v/v for 15 h.
N.B. for low protein concentration the destaining time might be too long. In such cases, shorter destaining times may optimize the procedure resulting in greater sensitivity.
7. Equilibrate the gel in water for 10 min, repeat once and scan.
Size Marker • © 2010 AppliChem
Prod. No.
Acrylamide Molecular biology grade
Bisacrylamide Molecular biology grade
Agarose Basic
Agarose low EEO (Agarose Standard)
Agarose MP
Loading buffer DNA I
Loading buffer DNA II
Loading buffer DNA IV (for Agarose gels)
TAE buffer (50X)
TAE buffer (10X)
TBE buffer (10X)
A3812
A3636
A8963
A2114
A1091
A3144
A2571
A3481
A1691
A1416
A0972
Transfer membranes
Prod. No.
Reprobe Nitrocellulose supported 0.22 μm Transfer membrane
Reprobe Nitrocellulose supported 0.45 μm Transfer membrane
Pure Nitrocellulose unsupported 0.22 μm Transfer membrane
Pure Nitrocellulose unsupported 0.45 μm Transfer membrane
Pure Nylon Neutral Transfer membrane 0.22 μm (30 cm x 3 m)
Pure Nylon Neutral Transfer membrane 0.45 μm
Reprobe Nylon Positively charged Transfer membrane 0.45 μm (30 cm x 3 m)
PVDF-Star Transfer membrane 0.45 μm
Other Related Products
A5237
A5242
A5250
A5239
A4399
A5248
A5255
A5243
Prod. No.
Chemiluminescence Detection Kits for Horseradish Peroxidase
Cheluminate-HRP PicoDetect
Cheluminate-HRP FemtoDetect
Cheluminate-HRP FemtoDetect Plus
Boric acid Molecular biology grade
EDTA disodium salt dihydrate Molecular biology grade
Tris ultrapure
A3417
A7807
A7879
A2940
A2937
A1086
related produc t s
Electrophoresis Reagents
Transfer Membranes
AppliChem supplies a range of transfer membranes designed
and tested speci­fically for RNA, DNA and protein analysis.
AppliChem also provides our customers with tried
and proven protocols developed to obtain consistent repro­ducible
and dependable results when used with AppliChem membranes.
22 protocols and all types of membranes – all from one source.
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