BCA (bicinchoninic acid) method

Workflow required to perform the BCA method:

• Protein purification process to determine yield and purity.
• Before gel electrophoresis, ensure equal protein loading.
• Before labeling or modifying proteins, make sure you are using the correct amount of protein.
• In cell culture studies, protein expression levels are determined.

Best Methods for BCA Testing

Standard BCA method

Best suited for routine applications where the protein concentration range is within the linear range of the assay and the sample number is moderate.

1. Sample preparation: First collect and prepare the protein sample to ensure that there are no interfering substances in the sample.
2. Preparing the BCA working reagent: This usually involves mixing a BCA solution with a copper(II) sulfate solution in specific proportions, usually provided by the assay kit manufacturer.
3. Mixing and Incubation: Protein samples are typically added to the BCA working reagent in a cuvette or test tube. Typically the mixture is incubated at 37°C for 30 minutes.
4. Measurement: After the incubation, the absorbance of each sample was measured using a spectrophotometer at a wavelength of 562 nm.
5. Analysis: The absorbance value is compared to that of a standard protein, usually bovine serum albumin (BSA), to determine the protein concentration of the sample.

Microplate BCA method

Ideal for high-throughput screening where many samples need to be analyzed simultaneously. It has high reagent usage efficiency and is often used in large research or industrial settings.

1. Sample Preparation: As with the standard assay, first prepare the protein sample.
2. BCA working reagent: Prepare the same reagent but adjust the volume to the microplate wells.
3. Aliquot and incubate: Use a multichannel pipette to aliquot protein samples and BCA working reagents into the wells of a 96-well plate. Typically incubate at 37°C for 30 minutes.
4. Measurement: Measure absorbance at a wavelength of 562 nm using a microplate reader.
5. Analysis: As with standard methods, protein concentration was determined using a standard curve.

Modified BCA method for detergent-containing samples

Use specifically for samples containing detergents as detergents can interfere with the BCA reaction. This is commonly seen when extracting proteins from certain cell types or tissues, or when studying proteins embedded in membranes.

1. Sample Preparation: Extract proteins from the sample, paying attention to detergent concentration.
2. BCA working reagent: Prepared according to standard detection methods.
3. Add detergent compatibility reagents: These reagents are usually provided in specialized BCA kits and are added to eliminate the interfering effects of detergents.
4. Mixing and Incubation: As with standard assays, sample and working reagents are mixed and incubated.
5. Measurement and Analysis: Measure absorbance using a spectrophotometer and determine protein concentration from a standard curve.

How to automate the BCA method:

• Liquid Handling Workstations: These machines are designed to automatically pipette liquids into designated containers, ensuring accuracy and consistency.
• Integrated systems: Some platforms integrate the entire process from sample addition to absorbance reading, simplifying workflow.
• Software: Automation software is available to set up the protocol, ensuring that each assay is performed the same way.

Advantages of the automated BCA method compared to manual pipetting:

• Consistency: Automation reduces variability by ensuring each step is performed the same way every time.
• Efficiency: Automated systems can process multiple samples simultaneously, speeding up processing.
• Accuracy: Automated pipetting reduces the risk of volume errors and cross-contamination.
• Data integrity: Automated systems often come with integrated data management solutions to ensure results are recorded and stored correctly.