|BCA Protein Assay Reagent A||250 ml|
|BCA Protein Assay Reagent B||25 ml|
|BCA Protein Assay Albumin Standard||10 units|
|Compatibility Reagent||48 ea|
|Reconstitution Buffer||15 ml|
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.
Additional Required Materials:
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).
|Vial||Diluent/Sample Buffer Volume (µl)||BSA Source and Volume (µl)||Concentration (µg/ml)|
|A||0||200 of stock||2,000|
|B||66||200 of stock||1,500|
|C||100||100 of vial A||1,000|
|D||100||100 of vial B||750|
|E||100||100 of vial C||500|
|F||100||100 of vial E||250|
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.
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.
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.
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.
Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) curve produces more accurate fit than a linear curve.
posted June 2020
Protocol Id: 2104
Bicinchoninic Acid (BCA) is capable of forming an intense purple complex with cuprous ion, Cu1+, in an alkaline environment. Cu1h+ is produced from the reaction of protein with alkaline Cu2+. The resulting reaction and color produced is the basis for a common protein quantification method capable of measuring protein concentration over a wide range. Increasing protein concentrations produce proportionally deeper colors. The BCA protein assay demonstrates higher tolerances towards common interfering substances, such as nonionic detergents and buffer salts, than the Lowry technique (1).
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