Workflows where BCA Assay is needed:
- Protein purification processes to determine the yield and purity.
- Before gel electrophoresis to ensure equal protein loading.
- Prior to protein labeling or modification to ensure the correct amount of protein is used.
- In cell culture studies to determine protein expression levels.
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.
- Sample Preparation: Begin by collecting and preparing protein samples, ensuring they’re free of interfering substances.
- 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.
- 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.
- Measurement: Once incubation is completed, measure the absorbance of each sample at 562 nm using a spectrophotometer.
- 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.
- Sample Preparation: As with the standard assay, start by preparing protein samples.
- BCA Working Reagent: The same reagent is prepared, but the volumes are adjusted for microplate wells.
- 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.
- Measurement: A microplate reader is used to measure the absorbance at 562 nm.
- 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.
- Sample Preparation: Extract proteins from samples, ensuring to note the concentration of detergents.
- BCA Working Reagent: Prepared as in the standard assay.
- Addition of Detergent Compatibility Reagents: These reagents, often provided in specialized BCA kits, are added to negate the interfering effects of detergents.
- Mixing & Incubation: Like the standard assay, mix the samples and working reagent and incubate.
- Measurement & Analysis: Use a spectrophotometer to measure absorbance and determine protein concentration via a standard curve.