Protein Determination by the Bicinchoninic Acid (BCA) Method

1. OBJECTIVE

To provide a standard procedure for the quantitative determination of total protein concentration of a solution by the BCA method.

2. SCOPE

This protocol applies to all regulated products requiring protein analysis by the BCA method.

3. DISCUSSION

Proteins reduce alkaline Cu (II) to Cu (I) in a concentration dependent manner. Bicinchoninic Acid is a highly specific chromagenic reagent for Cu (I) forming a complex with an absorbance maximum at 562 nm. Because of this property, the resultant absorbance at 562 nm is directly proportional to the protein concentration. Bovine serum albumin is used as a protein standard.

4. DEFINITIONS

N/A

5. RESPONSIBILITIES

N/A

6. PROCEDURE

6.1 Reagent

6.1.1 BicinchoninicAcid Solution, Product No. B9643.

6.1.2 Copper (II) Sulfate Pentahydrate 4% Solution, Product No. C2284.

6.1.3 Protein Standard Solution, 1.0 mg/mL Bovine Serum Albumin (BSA), Product No. P0914.

6.1.4 Test Samples

NOTE: Other BSA may be used as a standard. However, if dilution to 1 mg/mL is required, confirm the protein concentration by absorbance at 280 nm using E^1%₂₈₀ = 6.67. Samples should be between 0.2 - 10 mg/mL protein to remain within the linear range of this assay. Dilution with water may be required to fall within the range.

6.2 Materials

6.2.1 Spectrophotometer, Uvikon 943, or equivalent.

6.2.2 Pipet/tips

6.2.3 Test tubes

6.2.4 Test tube rack

6.2.5 Disposable cuvets, Product No. C5416, or equivalent.

6.2.6 Incubators, Imperial III, or equivalent.

6.3 Safety Precautions

Refer to Material Safety Data Sheets (MSDS) for chemical hazards and appropriate handling precautions.

6.4 Method

6.4.1 Prepare the protein determination reagent by adding I mL of Copper (KK) Sulfate Pentahyrate 4% Solution (C2284) to 49 mL of the Bicinchoninic Acid Solution (B9643).

6.4.2 Quantitatively add the indicated amounts of water and protein standard to the indicated tubes.

Tube	mL Water	mL Protein Standard
1	0.5	0
2	0.3	0.2
3	0.2	0.3
4	0.1	0.4
5	0.0	0.5

6.4.3 Prepare reaction tubes as follows: Samples are to be prepared in duplicate.

Tube	mL Protein Standard	mL Sample or Control	mg/mL Protein	mL Protein DeterminationReagent
1	1	-	0	2.0
2	0.1	-	0.4	2.0
3	0.1	-	0.6	2.0
4	0.1	-	0.8	2.0
5	0.1	-	1.0	2.0
6	-	0.1	?	2.0
7	-	0.1	?	2.0

6.4.4 Vortex each tube to ensure adequate mixing.

6.4.5 Incubate tubes at 37 °C for 30 minutes.

6.4.6 Allow tubes to cool to room temperature before measuring the absorbance.

6.4.7 Blank the spectrophotometer versus water. Measure the absorbance of the standards (tubes 1-6) at 562 nm.

6.4.8 Use the known protein concentration of the standards to create a standard curve. If data points show significant deviation from the standard curve line, consult departmental supervision. The slope of the line should be approximately 1.0.

6.4.9 Read the absorbance of the samples at 562 nm. Use the standard curve to calculate the protein concentration in mg/mL..

6.4.10 For final protein concentration, multiply by any dilution factor which was required to bring the concentration within the linear range of the assay. Average the results of your sample for reporting.

6.4.11 Complete the appropriate worksheet.