BCA Protein Assay Kit

IC-7991

Background

BCA Protein Assay Kit is a ready-to-use detergent- compatible Western blot related total protein analysis reagent used for the quick determination of total protein concentration by measuring absorbance at 562 nm and comparing to a protein standard absorption vs. concentration curve, according to Smith. The protein quantification process can be finished in 45 minutes.
The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantization of total protein. This method combines the well- known reduction of Cu2+ to Cu1+ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 ¦Ìg/ml).
The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA. Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color producing functional groups.
Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown before the concentration of each unknown is determined based on the standard curve. If precise quantization of an unknown protein is required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples. Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1 ml) of protein sample; however, because it uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized.

Protocol

A. Test Tube Procedure

1.Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml of BCA Reagent A with 1 ml BCA Reagent B.

Note: Use the following formula to determine the total volume of working reagent required. (# standards+ # unknowns) x (# replicates) x (volume of working reagent per sample)= total volume of working reagent required.

2.Follow Table 1 to prepare a fresh set of standards. (Dilute Albumin (BSA) Standards with 0.9% NaCl or PBS)

Table 1: Preparation of Albumin (BSA) Standards

Tube Number

Volume of Diluent (¦Ìl)

Volume of BSA (¦Ìl)

Final BSA Concentration (¦Ìg/ml)

A

0 ¦Ìl

900 ¦Ìl of 2 mg/ml Stock

2000 ¦Ìg/ml

B

100 ¦Ìl

300 ¦Ìl of tube A

1500 ¦Ìg/ml

C

300 ¦Ìl

300 ¦Ìl of tube A

1000 ¦Ìg/ml

D

200 ¦Ìl

200 ¦Ìl of tube B

750 ¦Ìg/ml

E

300 ¦Ìl

300 ¦Ìl of tube C

500 ¦Ìg/ml

F

300 ¦Ìl

300 ¦Ìl of tube E

250 ¦Ìg/ml

G

300 ¦Ìl

300 ¦Ìl of tube F

125 ¦Ìg/ml

H

400 ¦Ìl

100 ¦Ìl of tube G

25 ¦Ìg/ml

I

300 ¦Ìl

0

0 (blank)

3.Add 0.1 ml of each standard and protein samples into separate labeled test tubes.

4.Add 2 ml of BCA working reagent to each tube and mix well.

5.Incubate at 37¡ãC for 30 minutes.

Note: Increasing the incubation time and temperature can increase the net 562 nm absorbance for each test and decreases both minimum detection level and test range of the kit.

6.Cool all tubes to room temperature (RT).

7.Set the wavelength of spectrophotometer at OD 562 nm. Calibrate the instrument to zero by using water. Subsequently, measure the absorbance of all samples within 10 minutes.

Note: Color development continues even after cooling to RT. However, the subsequent development at RT is too weak to produce significant error if all absorbance measurements are made within 10 minute.

8.Subtract OD562 of Blank from all readings.

9.Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

B. Microplate Procedure

1. Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml of BCA Reagent A with 1 ml BCA Reagent B.

Note: Use the following formula to determine the total volume of working reagent required. (# standards+ # unknowns) x (# replicates) x (volume of working reagent per sample)= total volume of working reagent required.

2.Follow Table 2 to prepare a fresh set of standards. (Dilute Albumin (BSA) Standards with 0.9% NaCl or PBS)

Table 2: Preparation of Albumin (BSA) Standards

Tube Number

Volume of Diluent(¦Ìl)

Volume of BSA (¦Ìl)

Final BSA Concentration (¦Ìg/ml)

A

0 ¦Ìl

200 ¦Ìl of 2 mg/ml Stock

2000 ¦Ìg/ml

B

30 ¦Ìl

90 ¦Ìl of tube A

1500 ¦Ìg/ml

C

60 ¦Ìl

60 ¦Ìl of tube A

1000 ¦Ìg/ml

D

60 ¦Ìl

60 ¦Ìl of tube B

750 ¦Ìg/ml

E

60 ¦Ìl

60 ¦Ìl of tube C

500 ¦Ìg/ml

F

60 ¦Ìl

60 ¦Ìl of tube E

250 ¦Ìg/ml

G

60 ¦Ìl

60 ¦Ìl of tube F

125 ¦Ìg/ml

H

100 ¦Ìl

25 ¦Ìl of tube G

25 ¦Ìg/ml

I

60 ¦Ìl

0

0 (blank)

3. Add 25 ¦Ìl of each standard and protein samples into separate microplate wells.

4. Add 200 ¦Ìl of BCA working reagent to each well and mix well.

5. Seal plates and incubate at 37¡ãC for 30 minutes.

6. Cool plate to room temperature (RT).

7. Measure the absorbance at 562 nm on a plate reader within 10 minutes.

8. Subtract OD₅₆₂ of Blank from all readings. Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

Important product information

1. If this kit is received or stored cold, a precipitate may form in Reagent A or Reagent B. To dissolve the precipitate, warm the solution slowly at 37¡ãC while mixing or microwave for a few seconds. Discard the kit if it is contaminated by bacteria

2. If interference caused by reducing substances or metal- chelating substances contained in the sample remains, Bradford Assay Kit is recommended.

3. It is recommended that the standard of different concentrations and samples be assayed in duplicate. Standard curve should be plotted for each assay.

4. Newly formed green turbidity will disappear after mixing Reagent A and Reagent B thoroughly. It will not affect the performance.

5. Assayed sample amount will be reduced while using spectrophotometer to detect protein concentration. When using 37¡ã C incubator, prevent the influence of water evaporation.

6. Several ways for eliminating or minimizing the effects of interfering substances:

• Remove interfering substances by dialysis or gel filtration.

• Dilute the sample until substances no longer interfere.

• Since detergents of high concentrations also affect the results, precipitate the proteins in the sample with trichloroacetic acid (TCA).

7.Avoid using substances including reducing substances, chelating agents, strong acid and alkali since they interfere with protein estimation even at very low concentration.

Order Information

Cata.No.

Components

Price($£©

Price(€)

Price(£¤/CNY£©

IC-7991

250 tests

50.00

60.00

500.00

IC-7991

500 tests

70.00

84.00

700.00

IC-7991

2500 tests

240.00

288.00

2400.00

Applications

Western blotting;Protein expression assays;Protein profiling and characterization;Protein quantitation assays.

Shipping

Ship with blue ice.

Storage Conditions

Reagents A and B are valid for one year at room temperature. Protein store at standard room temperature storage valid for one month; Store at 4¡ãC valid for one year; Store at -20¡ãC can be stored for a long time.

Usage

For Research Use Only. Not For Use in Humans.