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BCA Protein Assay

Preparation of Diluted BSA Standards

Prepare the fresh set of protein standards by diluting a 2.0mg/ml BSA stock standard (stock), preferably in the same diluent as your sample. For a list of standard dilution, see table: Preparation of the Diluted BSA Standards. The BSA standard (1ml ampule of the 2.0mg/ml) is sufficient to prepare a set of diluted standards for either working range. There will be sufficient volume for three replications of each diluted BSA standard if so desired.

Preparation of the Diluted BSA Standards:

Standard microwell plate protocol

Vol. of BSA to add Vol. of Dilute to add Final BSA concentration
300l of (stock) 0l 2000g/ml
375l of (stock) 125l 1500g/ml
325l of (stock) 325l 1000g/ml
175l of (A) 175l 750g/ml
325l of (B) 325l 500g/ml
325l of (D) 325l 250g/ml
325l of (E) 325l 125g/ml
100l of (F) 400l 25g/ml

Working range = 2-2000g/ml

Preparation of the BCA Working Reagent (WR)

To prepare the WR, mix 50 parts of BSA reagent A with 1 part of BSA reagent B. When BSA reagent B is initially added to BCA reagent A, a turbidity is observed that quickly disappears upon mixing to yield a clear grreen WR. Prepare sufficient volume of WR based upon the number of tests to be done. Each test tube sample to be done requires 2.0ml of the WR, while the microwell plate samples require only 200l. The WR is stable for at least 1 day when stored in a closed container at room temperature.

The Microwell Plate Protocol

Sample to WR ratio is 1:9.

  1. Pipet 25l of each standard or unknown sample into the appropriate microwell plate wells. Use 25l of the diluent for the blank wells.
  2. Add 200l of the WR to each well, and mix the plate's wells on a shaker for 30 seconds.
  3. Cover the plate and incubate for 30 minutes.
  4. After incubation, cool the plate to room temperature.
  5. Measure the absorbance at or near 562nm on a plate reader.
  6. Subtract the average A(562) reading for the blanks from the A(562) reading for each standard or unknown sample.
  7. Prepare a standard curve by plotting the average blank corrected A(562) reading for each BSA standard vs. its concentration in g/ml. Using the standard curve, determine the protein concentration for each unknown sample.

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This document was created for the neurogenetics laboratory run by P. H. Reddy, Ph.D. in the NSI of OHSU. Page last updated: 18 Sept 2001.