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BCA (Bicinchoninic Acid) Assay

A colorimetric assay used to determine the concentration of proteins in a solution. The primary purpose of the BCA Assay is to quantify the amount of protein present in a sample, which is essential for various downstream applications in molecular biology and proteomics.

Workflows where BCA Assay is needed:


Top Methods for BCA Assay

Standard BCA Assay

Best suited for routine applications where the protein concentration range is within the assay’s linear range, and the number of samples is moderate.


  1. Sample Preparation: Begin by collecting and preparing protein samples, ensuring they’re free of interfering substances.
  2. Preparation of BCA Working Reagent: This usually involves mixing BCA solution with copper(II) sulfate solution in a specific ratio, often provided by the assay kit manufacturer.
  3. Mixing & Incubation: Add the protein samples to the BCA working reagent, usually in a cuvette or a test tube. Incubate the mixture, typically at 37°C for 30 minutes.
  4. Measurement: Once incubation is completed, measure the absorbance of each sample at 562 nm using a spectrophotometer.
  5. Analysis: Compare the absorbance values with those of a standard protein, commonly bovine serum albumin (BSA), to determine the protein concentration of your samples.


Microplate BCA Assay

Ideal for high-throughput screening, where many samples need to be analyzed simultaneously. It’s efficient in terms of reagent usage and is often used in larger research studies or industrial settings.


  1. Sample Preparation: As with the standard assay, start by preparing protein samples.
  2. BCA Working Reagent: The same reagent is prepared, but the volumes are adjusted for microplate wells.
  3. Dispensing & Incubation: Using multi-channel pipettes, dispense the protein samples and BCA working reagent into the wells of a 96-well plate. Incubate, often at 37°C for 30 minutes.
  4. Measurement: A microplate reader is used to measure the absorbance at 562 nm.
  5. Analysis: As with the standard method, a standard curve is used to determine protein concentrations.


Modified BCA Assay for Detergent Samples

Specifically used when samples contain detergents, which can interfere with the BCA reaction. This is common in protein extraction from certain cell types or tissues or when studying proteins embedded in membranes.


  1. Sample Preparation: Extract proteins from samples, ensuring to note the concentration of detergents.
  2. BCA Working Reagent: Prepared as in the standard assay.
  3. Addition of Detergent Compatibility Reagents: These reagents, often provided in specialized BCA kits, are added to negate the interfering effects of detergents.
  4. Mixing & Incubation: Like the standard assay, mix the samples and working reagent and incubate.
  5. Measurement & Analysis: Use a spectrophotometer to measure absorbance and determine protein concentration via a standard curve.

BCA Assay has never been easier

The OT-2 is a bench-top liquid handler designed to be accessible and flexible enough to automate many common applications.

How to Automate the BCA Assay process:


Benefits of Automation over Manual Pipetting for BCA Assay

Protocol Spotlights

Opentrons helps you automate BCA Assay with open-source protocols for the OT-2 and Opentrons Flex


Want to know more?

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