Total Protein Assay Kit Manual

BIOO LIFE SCIENCE PRODUCTS
Total Protein
Assay Kit Manual
Catalog #: 5616-01
©BIOO Scientific Corp. • 2014
TABLE OF CONTENTS
GENERAL INFORMATION .......................................................................................... 1
Product Description ............................................................................................................. 1
Introduction .............................................................................................................................. 1
Procedure Overview ............................................................................................................. 1
Kit Contents, Storage and Shelf Life ............................................................................ 2
Required Materials Not Provided With the Kit ....................................................... 2
Warnings and Precautions ................................................................................................ 2
SAMPLE PREPARATION .............................................................................................. 3
TOTAL PROTEIN DETERMINATION TEST PROTOCOL ..................................... 3
Set up ........................................................................................................................................... 3
Reagent Preparation ............................................................................................................ 3
Sample Test Procedure ........................................................................................................ 4
Total Protein Concentration Calculation .................................................................. 4
MaxDiscovery™ Total Protein Assay Kit is intended for laboratory use only, unless otherwise indicated. This product is
NOT for clinical diagnostic use. MaxDiscovery is a Trademark of Bioo Scientific Corporation.
MaxDiscovery™ Total Protein Assay Manual – 5616-01
GENERAL INFORMATION
GENERAL INFORMATION
Product Description
The MaxDiscovery™ Total Protein Assay Kit is a plate-based colorimetric enzymatic assay for
the determination of total protein in serum samples.
The kit uses a spectrophotometric assay to detect total protein directly from serum samples,
enabling researchers to detect total protein levels in animal serum and other tissue matrices
such as urine and ascites. The unique features of the kit are:



High sensitivity and low detection limit (0.5 g/dL).
A rapid (10 minute) and robust enzyme-based assay which does not require expensive
instrumentation.
High reproducibility.
Introduction
Proteins form the major portion of the macromolecular solutes in the blood. Protein in the
plasma is primarily comprised of albumin and globulin. Changes in blood protein levels are
associated with various disease states. Elevated levels of total protein in plasma have been
strongly associated with a variety of diseases involving the liver, kidney, or bone marrow, as
well as other metabolic or nutritional disorders. Low serum protein concentrations can be
symptoms of overhydration, kidney damage, severe burns, or failure of protein synthesis
(starvation, protein malnutrition, or liver cell damage).
The MaxDiscovery Total Protein Assay Kit is a simple, direct and automation-compatible
method for measuring total protein levels in serum samples. This method, commonly referred
to as the Biuret assay, is based on the interaction of cupric ion (Cu2+) with protein in an alkaline
solution.
Protein + Cu2+ → blue-violet complex
The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the
protein concentration. This kit provides direct determination of total protein in serum, plasma,
and other fluid samples.
The kit contains sufficient materials to rapidly test 42 serum samples in duplicate. It also comes
with a control solution containing a total protein standard which can be used to calibrate the
assay.
Procedure Overview
After preparing the sera, the assay is performed by adding Total Protein Reagent into
microplate wells containing 6 µL sera. After a brief incubation, the absorbance of each well at
540 nm is then measured using a plate reader. The concentration of total protein in each
sample is then directly determined from the 540 nm absorbance.
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MaxDiscovery™ Total Protein Assay Manual – 5616-01
Kit Contents, Storage and Shelf Life
The MaxDiscovery™ Total Protein Assay Kit has the capacity for 96 determinations or testing
of 42 samples in duplicate (using 12 wells for standards). Store the kit at 4°C. The shelf life is 6
months after receipt when the kit is properly stored.
Kit Contents
Microtiter Plate
Total Protein Standard
Standard Diluent
Total Protein Reagent
Amount
1
1 vial
4 mL
30 mL
Storage
4°C – 30 °C
4 ºC
4°C
4 ºC
Required Materials Not Provided With the Kit





Microtiter plate reader (540 nm).
Centrifuge to prepare serum samples.
Deionized or distilled water.
1.5 mL microfuge tubes.
Multichannel pipet or repeating pipettor (recommended but not required).
Warnings and Precautions
BIOO strongly recommends that you read the following warnings and precautions to
ensure your full awareness of the techniques and other details you should pay close
attention to when running the assays. Periodically, optimizations and revisions are made to
the kit and manual. Therefore, it is important to follow the protocol that comes with the kit. If
you need further assistance, you may contact your local distributor, or BIOO at
[email protected].




Add standards to plate only in order from low concentration to high concentration, as this
will minimize the risk of compromising the standard curve.
Anticoagulants such as fluoride and oxalate will result in false low values.
The assay is not influenced by hemoglobin values up to 200 mg/dl or by bilirubin levels up
to 10 mg/dl.
Interference from grossly hemolyzed specimens is correctable by use of a serum/plasma
blank.
BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its
products are made are of high quality. There is no warranty of merchantability of this product, or of the
fitness of the product for any purpose. BIOO shall not be liable for any damages, including special or
consequential damage, or expense arising directly or indirectly from the use of this product.
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MaxDiscovery™ Total Protein Assay Manual – 5616-01
SAMPLE PREPARATION
SAMPLE PREPARATION
1. Carefully prepare at least 20 µL of sera using standard production procedure (if
determinations are performed in singlet then 10 µL is sufficient). Avoid hemolysis, as it
may release erythrocyte total protein into the serum.
2. Total protein in serum is reported stable for 7 days when stored at 4°C, and 72 hours at
room temperature (23-25 °C)
Note:
1. Samples with values above 10 g/dL should be diluted 1:1 with saline (0.9% NaCl) and
re-tested. Multiply results by two.
2. Grossly lipemic serums require a sample blank. Add 5 µL of sample to 250 µL saline, mix,
and read the absorbance against water. Subtract this reading value from the absorbance
of each serum sample to obtain the corrected reading.
CHOLESTEROL
TEST PROTOCOL
TOTALDETERMINATION
PROTEIN DETECTION
TEST PROTOCOL
Set up
Prewarm reagents to 37°C for 1 hour. Turn on the plate reader and allow the lamp to warm up. Set
the temperature of the plate reader to 37°C before reading. Adjust the wavelength of the plate
reader to 540 nm.
Be sure that Total Protein Reagent is prewarmed to 37°C before use.
Reagent Preparation

Preparation of Total Protein Standard
To reconstitute the Total Protein Standard, add exactly 3 mL of Standard Diluent to the Total Protein
Standard. Mix by swirling or inverting the bottle 10 - 12 times. Allow contents to dissolve for 30
minutes at room temperature.
IMPORTANT: The reconstituted Total Protein Standard can be left at room
temperature for short periods (up to 4 hours) prior to use. Between uses, the
reconstituted Total Protein Standard should be stored frozen at -20 C (for up to 180
days). Divide the reconstituted Standard into smaller aliquots to avoid repeated
freeze/thaw cycles.
Preparation of Total Protein Control dilutions for Standard Curve
1. Label 6 microfuge tubes: 1, 2, 3, 4, 5, 6.
2. Dilute the Total Protein Standard using Standard Diluent as described in the table below.
After dilution, briefly mix each tube.
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MaxDiscovery™ Total Protein Assay Manual – 5616-01
Tube #
1
2
3
4
5
6
Volume Total Protein
Standard (6.3 g/dL)
0 µL
15 µL
30 µL
45 µL
60 µL
90 µL
Volume Distilled Water
90 µL
75 µL
60 µL
45 µL
30 µL
0 µL
Total protein
Concentration (g/dL)
0
1.05
2.1
3.15
4.2
6.3
3. Use 6 µL of each diluted standard per reaction (see below).
Sample Test Procedure
1.
2.
3.
4.
Add 6 µL of each sample or standard (in duplicate) to the microplate wells.
Add 300 µL of reagent mix to the wells.
Incubate at 37C for 10 minutes.
Measure the absorbance of each sample at 540 nm.
Total Protein Concentration Calculation
There is a linear relationship between the concentration of total protein in the sample and
absorbance at 540 nm. Therefore, a standard curve used to calculate the total protein
concentration in sera samples can be constructed by plotting the mean corrected absorbance
values for each of the diluted total protein standards as a function of total protein concentration.
The standard curve provides a reference for the linear range of the assay.
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MaxDiscovery™ Total Protein Assay Manual – 5616-01
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Bioo Scientific Corporation
7050 Burleson Rd
Austin, TX 78744 USA
Tel: 1.888.208.2246
Fax: (512) 707-8122
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[email protected]
[email protected]
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