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QuantiChromTM Protein Assay Kit
(QCPR-500)
Bradford Colorimetric Protein
Determination at 595 nm
DESCRIPTION
The
protein is known as the "building blocks of life" and is
one of the most important macromolecules in life
science. Proteins are polypeptides made up of amino
acids and play various key roles in all aspects of
biology. Protein quantitation is a very common practice
for life scientists. Simple, direct and automation-ready
procedures for measuring protein concentration are very
desirable. BioAssay Systems' QuantiChromTM protein assay
kit is based on an improved Coomassie Blue G method. The
dye forms a blue complex specifically with protein, and
the intensity of color, measured at 595nm, is directly
proportional to the protein concentration in the sample.
The optimized formulation substantially reduces
interference by substances in the raw samples and
exhibits increased sensitivity towards peptides.
APPLICATIONS
Direct
Assays:
total
protein concentration.
KEY
FEATURES
Sensitive
and accurate.
Use 10 μL samples. Detection range 0.06 – 1.0
mg /mL protein in 96-well plate assay.
Simple and high-throughput.
The “mix-and-read” procedure involves addition of a
single working reagent and reading the optical density.
Can be readily automated as a high-throughput assay in
96-well plates for thousands of samples per day.
Low interference. Glucose,
Tris, vitamins, and amino acids, DNA, RNA, salts, EDTA
(< 12 mM), phenol (< 50 mM), urea (< 0.6 M), Triton (<
0.1%) and SDS (< 0.1% SDS) do not interfere in the
assay.
Versatility: assays can be
executed in 96-well plate or cuvet.
KIT
CONTENTS (500 tests in 96-well plates)
Reagent:
20 mL 5 x concentrate
Protein
standard: 1 mL 1.0 mg/mL BSA
Storage
conditions.
The kit is shipped at room temperature. Store the
reagent at room temperature and standard at -20°C,
respectively. Shelf life of at least 6 months (see
expiry dates on labels).
Precautions: reagents are for research use only.
Normal precautions for laboratory reagents should be
exercised while using the reagents. Please refer to
Material Safety Data Sheet for detailed information.
PROCEDURES
Reagent
Preparation: Prepare enough working reagent by
adding 1 vol of the 5 x Reagent to 5 vol of distilled
water. Bring reagent to room temperature before use.
Procedure using 96-well plate:
1. Dilute
standard as shown in the Table. Transfer 10 μL diluted
Standards and diluted sample in duplicate wells of a
clear bottom 96- well plate. Store diluted standards at
-20°C for future use.
2. Add 200 μL working reagent and tap lightly to mix.
3. Measure OD at 570-630nm (peak 595nm).
Procedure using cuvette:
1. Prepare standards as in the 96-well plate assay.
Transfer 50 μL diluted Standards and 50 μL samples to
cuvets.
2. Add 1000 μL working reagent and tap lightly to
mix.
3. Measure OD at 570-630nm (peak 595nm).
CALCULATION
Subtract blank OD (water, #8) from the standard OD
values and plot the OD against standard concentrations.
Use the standard curve to determine the sample protein
concentration.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipeting devices and accessories.
Procedure using 96-well plate:
Blank 96-well plates (e.g. Corning Costar). Plate
reader for 96-well plate.
Procedure using cuvette:
Cuvets and spectrophotometer.
GENERAL CONSIDERATIONS
If protein concentration is > 1 mg/mL, dilute samples
in distilled water, and use OD values that lie within
the calibration curve to calculate the sample protein
concentration. Reading can be performed as soon as the
reagent and sample are mixed. High sensitivity can be
achieved by adding 50 μL sample to 200 μL Reagent
(detection range 3 – 200 μg/mL).
LITERATURE
1. Bradford, M. (1976). A rapid and
sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72: 248-354.
2. Friedenauer, S. and Berlet, H.H.
(1989). Sensitivity and variability of the Bradford
protein assay in the presence of detergents. Anal.
Biochem. 178: 263-268.
3. Stoscheck, C. M. (1990). Increased
uniformity in the response of the Coomassie Blue G
protein assay to different proteins. Anal. Biochem. 184:
111-116.
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