Quantitation of Protein:Bicinchoninic Acid (BCA) Method
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【 Purpose 】
Master the BCA method and procedure to quantitate protein concentration
【 Principle 】
BCA method is a modification of Lowry method. The BCA technique offers manipulative simplifications, more tolerance toward interfering substances, greater working reagent stability, increased sensitivity (the minimum detection can be 0.5μg/ml), and greater protocol flexibility when compared to the standard Lowry assay.
The peptide bond of amino acids can bind with Cu 2+ to form a complex in the basic solution and then the Cu 2+ is deoxidized to Cu + . Bicinchoninic acid and its sodium salt are water-soluble.
In the alkaline environment, it can bind with Cu 2+ to form a dark purple compound, which has strong absorbance maximum at 562nm wavelength. Because the brightness is coordinated with the concentration of protein, we can use matching method to determine protein concentration.
【 Materials 】
1.Apparatus
721-spectrophotometer, Constant temperature water bath tank, Pipettes, Test tubes and test tube shelf
2.Reagent:
(1) BCA reagent:
① Reagent A, 1L:Weigh 10g BCA (1%), 20g Na 2 CO 3• H 2 O (2%)，1.6g Na 2 C 4 H 4 O 6• 2H 2 O (0.16%)，4g NaOH (0.4%) ，9.5g NaHCO 3 (0.95) respectively. Add water to 1L, and then adjust the pH to 11.25 by NaOH or solid NaHCO 3
② Reagent B, 50ml: Take 2g CuSO 4 ·5H 2 O (4%), add distilled water to 50ml.
③ BCA Reagent: Take 50 allots Reagent A and 1 allots Reagent B, mix up. This reagent can be stable for a week.
(2) Standard protein solution: Weigh 40mg bovine serum albumin, dissolved in distilled water to 1000ml, so the concentration of standard protein solution is 400μg/ml.
(3)Sample solution:Make about 50μg/ml bovine serum albumin solution.
【 Procedures 】
1. Draw calibration curve
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
|
Standard protein solution (μl) Distilled water（μl） BCA reagent（ml） Protein concentration（μg） |
0 250 5 0 |
50 200 5 20 |
100 150 5 40 |
150 100 5 60 |
200 50 5 80 |
250 0 5 100 |
After adding those reagents, shake them up, keep standing for 30 minutes at 37℃, then wait till the temperature drops to the room temperature. Determine absorbance at 562nm, the first tube is contrast. Make absorbance-protein concentration calibration curve, while the bovine serum albumin concentration is x-axis, the absorbance is y-axis.
2. Sample assay
Imbibe 250μl of sample solution truly to a clear and dry test tube, add 5ml of BCA reagent and shake up, incubate at 37℃ for 30 minutes. Use the first tube as contrast, and then make color matching at 595nm. Record the absorbance.
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Results
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