BCA Protein Assay Kit #7780

BCA Protein Assay Protocol

Important: This BCA Protein Assay Kit is compatible and has been thoroughly tested and approved to work with Cell Lysis Buffer (10X) #9803, RIPA Buffer (10X) #9806, and PathScan^® Sandwich ELISA Lysis Buffer (1X) #7018 when each is used at 1X. The compatibility of this assay has not been evaluated on any other buffers.

A. Solutions and Reagents

Kit Contents:

• BCA Reagent A: 250 ml (sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartarate in 0.1 M sodium hydroxide).
• BCA Reagent B: 25 ml (4% cupric sulfate).
• Compatibility Reagent: 9.3 mg × 48 microtubes.
• Reconstitution Buffer: 15 ml.
• Albumin Standard (2 mg/ml): 10 × 1 ml ampules of bovine serum albumin (BSA) in 0.9% NaCl and 0.05% sodium azide.

Additional Required Materials:

• 96-well plate compatible with absorbance measuring plate readers (such as Costar #3797)
• Adjustable volume pipettes and disposable pipette tips
• An incubator set at 37ºC
• Plate reader capable of measuring absorbance at 562 nm

B. Standard Preparation

Dilute the contents of one Albumin Standard (BSA) ampule into several microcentrifuge tubes, using the same buffer as the unknown sample(s). Use the following table as a guide to prepare a set of standards (assay range = 125-2,000ug/ml).

Note: Do not discard any unused, undiluted BSA standard (2 mg/ml). BSA standard can be stored in a microcentrifuge tube at 4°C for up to 2 weeks. Do not freeze.

C. Reagent Preparation

Working Reconstitution Buffer:

Dilute the Reconstitution Buffer 1:1 with dH₂O. Do not dilute the Reconstitution Buffer if the protein sample has a pH < 6.0 or if it contains EDTA or imidazole.

Compatibility Reagent Solution:

Puncture the foil covering on the Compatibility Reagent tube with a clean pipette tip. Add 100 µl of Working Reconstitution Buffer into the tube and dissolve by stirring the bottom of the tube and pipetting up and down 15-20 times. Store this solution for up to 8 hr at 4°C protected from light. Used microtubes may be cut and discarded from the unused microtubes. Return the unused microtubes to pouch containing the desiccant pack.

Note: 4 µl Compatibility Reagent Solution will be required for each sample assayed.

BCA Working Reagent (WR):

Use the following formula to determine the total volume of WR required:

(# controls + # standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR required.

Example: For three unknowns and two replicates of each sample:

(2 controls + 7 standards + 3 unknowns) × (2 replicates) × (0.26 ml) = 6.24 ml WR required.

To prepare the WR, mix 50 parts BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B). 6.24 ml WR = 6.12 ml Reagent A + 0.12 ml Reagent B

Note: When Reagent B is added to Reagent A, the solution appears turbid but yields a clear, green WR upon mixing.

D. Assay

Notes: Precision pipetting is essential. For best results, use 1-10 µl pipettes. Completely evacuate the tip of any fluid. Small errors when pipetting account for large errors when measuring the absorbance.

Add samples directly to the center of the well and avoid touching the sides of the well.

The same tip may be used within the same group of samples. Use a different tip for each sample when adding the Compatibility Reagent Solution.

If the protein sample has a pH < 5.0, dilute the sample 1:1 with Reconstitution Buffer.

1. Add 9 µl of Standard Control, Sample Control, Protein Standard, or unknown sample to individual wells on a microplate.
2. Add 4 µl Compatibility Reagent Solution to the sample in each well.
3. Cover plate and mix on a plate shaker at medium speed for 1 min and then incubate at 37°C for 15 min.
4. Add 260 µl WR to each well. Cover plate and mix on a plate shaker for 1 min. If the samples contain detergents, cover the plate after mixing. Incubate plate at 37°C for 30 min.
5. Cool plate at room temperature (RT) for 5 min.
6. Use the Standard Control as the blank. Measure the absorbance of the standards, unknown samples, and Sample Controls at 562 nm on a plate reader.

E. Data Analysis

Because the BCA Assay does not reach a true end point, color development will continue even after cooling to RT. The absorbance increases at a rate of ~0.25% per minute at RT.

1. Subtract the average 562 nm absorbance value of the Sample Control replicates from the 562 nm absorbance value of all unknown sample replicates.
2. Prepare a standard curve by plotting the average blank-corrected 562 nm value for each BSA standard against its concentration (µg/ml). Use the standard curve to determine the protein concentration of each unknown sample.

   Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) curve produces more accurate fit than a linear curve.