BCA Protein Assay

Measuring Protein with BCA Assay

Youssef Farhat, Written 7/16/12, Last updated 5/6/14

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Measuring the protein concentration of samples is essential for many protein assays, such as Western Blotting or Zymography.  This protocol makes use of Thermo Scientific’s BCA protein assay in a 96-well tissue culture plate format to measure the amount of protein in small volumes of sample.  I have found the assay to be fairly sensitive and reliable, however a few points should be noted:

  • As in many assays, samples are best measured in duplicate or triplicate to account for pipetting error
  • For the first time using the assay on a type of sample or lysis buffer, serially dilute some practice samples in order to establish a linear relationship between the known protein content and the measured signal
  • Prepare samples in cDNA tubes or other tubes that are made of a low-binding material
  • Bubbles can cause massive interference with this assay.  Be sure to pop bubbles before reading the plate and avoid making them as much as possible.
  • In my experience with cell lysates of tendon cells, one can expect to obtain about 0.4 ng of protein/cell.

Thermo Scientific’s BCA Protein Assay


  • BCA Protein Assay (Thermo Scientific, #23225)
  • Clear, flat-bottom 96-well plate (such as Falcon, #353072)
  • A plate reader that can measure absorbance at 560 nm
  • Typical lab supplies such as 1.5 mL Eppendorf tubes, cDNA strip tubes, pipettors, serological pipettes, micropipettors, multi-channel pipettor, reagent reservoirs, and pipette tips


1)       Samples should be homogenized, ready to measure, and kept on ice throughout the following steps.

2)       If not previously done, open a vial of BSA protein standard included with the kit and aliquot 120 µL into a number of 1.5 mL Eppendorf tubes.  Briefly centrifuge the droplets down to the bottom of the tubes and store the aliquots at -20°C.

3)       Prepare 1:1 serial dilutions of bovine serum albumin (BSA) standard provided by the kit (comes at 2 mg/mL).  This can be accomplished as follows:

  1. Pipette 55 µL of BSA standard into tube 1 in a set of cDNA strip tubes
  2. Pipette 55 µL of deionized water into tubes 2-8
  3. Serially dilute another 55 µL of BSA standard into tubes 2-7.  Do not dilute into tube 8 so that it can serve as a Blank for the assay.

4)       Diluting samples is sometimes necessary to reduce interference from reagents in the lysis buffer or to avoid using too much sample in the BCA assay.  This is how I typically dilute my samples:

  1. Transfer 20 µL of each sample to a new cDNA tube/Eppendorf tubes.
  2. Add 60 µL of deionized water to the samples (dilute by a factor of 4).

5)       Transfer 20 µL of samples and standards into the wells of a clear, flat-bottomed 96-well plate in duplicate or triplicate if possible.

6)       Prepare enough BCA assay reagents by combining 20 mL of Reagent A with 400 µL of Reagent B.  This is enough for a single 96-well plate; it can be scaled as necessary depending on the number of samples that need to be assayed.

7)       After mixing, add 180 µL of the BCA Reagent mixture to each well of the 96 well plate.

8)       Place in an incubator at 37 C for 30 min.  Remove and let it cool to room temperature.  Meanwhile, set the plate reader to read absorption at 560 nm.

9)       After reaching room temperature, place the plate onto the plate reader and measure absorption at 560 nm

10)   Retrieve the data, and using the BSA standard curve, interpolate the concentration of the samples.

11)   If your samples were diluted, be sure to scale back to the original concentration of the samples.  In the example dilution above, simply multiply the concentration by 4 to obtain the concentration of the original samples.

Citing this Protocol

Farhat, Y. “Measuring Protein with BCA Assay.” The Protocol Place. July 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com>

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