ZeptoMARK CeLyA Service Sample Results ZeptoMARK CeLyA Lysate Microarray Experimental Procedure

ZeptoMARK CeLyA Service Sample Results
ZeptoMARK CeLyA Lysate Microarray Experimental Procedure
In a ZeptoMARK CeLyA service study, your cell lysates or tissue lysates are
spotted on ZeptoMARK chips, after the total protein concentration has been
normalized. The ZeptoMARK CeLyA cell lysate arrays are then incubated with your choice of primary antibodies
against e.g. signaling proteins, one antibody per array. Utilizing a fluorescence labeled anti-species secondary
antibody, the amount of primary antibody bound in each lysate spot in the array is measured in the ZeptoREADER.
Duplicate arrays are analyzed for each primary antibody. The ultimate sensitivity of the ZeptoREADER technology
enables the researcher to detect minute amounts of bound antibody and thus to quantify relatively low abundant
proteins in the lysates. The microarray images are analyzed with the ZeptoView Pro software and the lysate spot
intensities are referenced to control spots resulting in Referenced Fluorescence Intensities (RFI). The RFI values for
each of your lysates assayed with each primary antibody are the raw data you will obtain from a CeLyA study. Bar
plot profiles, EC50 curves and fold change diagrams are simple visualizations of these data. - For details of the
experimental procedure please refer to the document “ZeptoMARK CeLyA Process”.
Expression and Activation Profiles
The measured relative abundance of a protein or a posttranslational protein modification against which the
primary antibody is directed is displayed as a bar plot
diagram. On the x-axis, 32 lysates spotted in one array
are listed. On the y-axis, the RFI value is plotted. The
error bars correspond to the variability between the
duplicate arrays incubated with the same primary
antibody.
The example in Figure 1 shows that e.g. in lysate 27 there
is 2 times more MAP kinase p44/42 present than in
lysate 26.
Figure 2 shows the results for the same lysates, this time
incubated a primary antibody specifically directed
against activated, phosphorylated MAP kinase p44/42.
The phosphorylation status of MAP kinase p44/42 in the
32 cell lysates can now be analyzed by comparing Fig. 1
and Fig. 2. For example: Whereas the level of total MAP
kinase p44/42 is identical in lysate 6 and 7, the level of
activated MAP kinase p44/42 in cell lysate 6 is twice as
high as in lysate 7. This indicates an increase in p44/42
MAPK signaling induced by for example the different
treatment of the cell cultures 6 and 7.
ZeptoMARK CeLyA Service Sample Results
EC50 Calculations
The efficacy of a drug (EC50 value) can be calculated
from results of ZeptoMARK CeLyA arrays representing
lysates treated with different concentrations of a
compound.
In the experiment of Figure 3, cell cultures of 2
different cell lines have been treated with 7 different
concentrations of a compound. The cells were lysed,
spotted in a ZeptoMARK CeLyA format and the effect
of the treatment on the target or other marker protein
quantified. Per definition, the EC50 value is the
compound concentration where half of the maximum
effect is reached.
EC50
1.0
0.8
0.6
Fig. 4
Cleaved caspase 3
Cyclin D1
P-Rb
P-Histone H3
beta-catenin
alpha-catenin
P-Stat3
P-SAPK/JNK
P-p38
1.2
P-Akt
1.4
P-Erk2
Fold signal changes (treated / control)
Biomarker Finding
In biomarker finding the relative abundance and
activation of proteins is investigated in order to identify
markers specific for healthy vs. disease states or
treated vs. control samples.
Fold changes as displayed in Fig. 4 are a useful way to
visualize differences between samples. In this example,
different expression and activation levels between
colorectal cancer tissue, chemotherapeutically treated
vs. control are visualized. Significantly less
phosphorylation is observed for Stress-activated
protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK).
The dashed lines indicate the significance threshold of
20% difference in RFI signal.
With the large number of samples that can efficiently
be screened with ZeptoMARK CeLyA lysate arrays it
becomes feasable to validate biomarkers on a large
statistical basis.