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Bradford – Protein Determination
Introduction
A rapid and accurate method for the estimation of protein concentration. The technique is simpler, faster than the Lowry method, and is subject to less interference. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (1976) 72, 248-254.
The Coomassie blue G250 dye appears to bind most readily to arginyl and lysyl residues of proteins (not to the free amino acids). This specificity can lead to variation in the response of the assay to different proteins, which is the main disadvantage of the method.
Reagents & Solution
Bovine Serum Albumine (BSA) 10X: 1mg/ml H2O. Keep –20C in aliquots.
BSA 1X (0.1mg/ml): dilute BSA 10X in the buffer of your sample.
Bradford solution from Bio Rad Protein Assay. Kep in dark at 4C. (Do not use directly from the bottle. Transfer the volume you are going to use to another recipient).
Mini: for 500µl determination in 1.5ml plastic tubes.
|
BSA 100 µg/ml (µl) |
H2O ((µl) |
Bradford (µl) |
total µg BSA |
|
0 |
400 |
100 |
0.0 |
|
10 |
390 |
100 |
1.0 |
|
20 |
380 |
100 |
2.0 |
|
35 |
375 |
100 |
3.5 |
|
50 |
350 |
100 |
5.0 |
|
X ml of sample |
up to 400 |
100 |
X |
Mix and wait 2min then read within 60 min at OD590nm. It is preferable to use disposable polystyrene microcuvettes that are discarded after a series of absorbance measurements. Plot OD590nm versus µg BSA standard. Try to use a sample concentration with OD590nm near the middle of the curve. Prepare triplicates of your sample, or use different points. That is, use several dilutions that you estimate will be inside the linearity of the curve, or prepare triplicates of your sample that you estimate to be in the middle of the curve. The linearity of the assay can be improved by plotting the ratio of absorbances at 595 and 465 nm.
Elisa plates: for 125µl determination in 96 microwell plates.
|
BSA 100 µg/ml (µl) |
H2O (µl) |
Bradford (µl) |
total µg BSA |
|
0 |
100 |
25 |
0.0 |
|
3 |
97 |
25 |
0.3 |
|
6 |
94 |
25 |
0.6 |
|
9 |
91 |
25 |
0.9 |
|
12 |
88 |
25 |
1.2 |
|
X ml of sample |
up to 100 |
25 |
X |
Mix and read immediately at OD590nm. Plot OD590nm vs. ug BSA. Try to use a sample volume with OD590nm near the middle of the curve.Prepare triplicates of your sample, or use different points.
Home made reagent:
Dissolve 100 mg of Coomassie blue G250 in 50 mL of 95% ethanol. Mix with 100 ml of 85% phosphoric acid and made up to 1 L with distilled water. Filter through Whatman No. 1 filter paper and then stored in an amber bottle at room temperature. It is stable for several weeks. Filter before use if there is any dye precipitation.
Effects of Common Reagents on the Bradford Assay
Absorbance at 600 nm
Compound |
Blank |
5 mg IgG |
|
Control |
0.005 |
0.264 |
|
0.02% SDS |
0.003 |
0.250 |
|
0.1% SDS |
0.042 |
0.059 |
|
0.1% Triton |
0.000 |
0.278 |
|
0.5% Triton |
0.051 |
0.311 |
|
1M 2-Mercaptoethanol |
0.006 |
0.273 |
|
1M Sucrose |
0.008 |
0.261 |
|
4M Urea |
0.008 |
0.261 |
|
4M NaCl |
0.015 |
0.207 |
|
Glycerol |
0.014 |
0.238 |
|
0.1M HEPES, pH 7.0 |
0.003 |
0.268 |
|
0.1M Tris, pH 7.5 |
0.008 |
0.261 |
|
0.1M Citrate, pH 5.0 |
0.015 |
0.249 |
|
10 mM EDTA |
0.007 |
0.235 |
|
M (NH 4) 2SO 4 |
0.002 |
0.269 |
Data were obtained by mixing 5 uL of sample with 5 uL of the specified compound before adding 200 uL of dye-reagent. According to Stoscheck, C. Increased uniformity in the response of the Coomassie blue protein assay to different proteins. Anal. Biochem. (1990) 184, 111-116.
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