BCA (bicinchoninic acid) uses copper reduction. It is compatible with most detergents, including SDS and Triton X-100, so it suits cell lysates prepared with RIPA or NP-40 buffer. Bradford dye-binding is faster and more sensitive at low concentrations, but detergents above approximately 0.1 percent inhibit it. Choose BCA for detergent-containing samples and Bradford for detergent-free, purified protein solutions.
Proteins
Protein Quantification Kits for BCA and Bradford Assays
Protein quantification kits for accurate, sample-matched BCA, Bradford, and Biuret assays
The right protein quantification assay gives you accurate total protein measurements that remain compatible with your sample matrix. This matters whether you are loading a gel, normalising an ELISA plate, or preparing a proteomics sample.
Three colorimetric formats cover most of your laboratory needs. BCA assays reduce cupric to cuprous ions in an alkaline environment, forming a purple complex measurable at 562 nm. They tolerate moderate detergent concentrations, so they suit your cell lysates prepared with RIPA or NP-40 buffers. Bradford assays bind Coomassie Brilliant Blue G-250 to protein and shift the dye absorbance from 465 nm to 595 nm. They deliver fast results, in five minutes, but are more susceptible to interference from detergents and basic buffers. Biuret assays detect peptide bonds directly in an alkaline copper solution, read around 540 to 560 nm. Biuret is simple and reagent-stable, but less sensitive than BCA or Bradford, so it suits your samples with higher protein concentrations.
BCA, Bradford, and Biuret formats are all available to you, validated for lysate and purified protein inputs. For GFP-tagged protein quantification independent of total protein, an anti-GFP antibody sandwich assay format gives you selective GFP detection in crude cell lysates without gel separation.
The assay follows the sample type and dynamic range.
1) Detergent-containing samples such as RIPA or NP-40 lysates suit BCA, working at 0.2 to 2 mg per ml, read at 562 nm after a 30-minute incubation at 37°C.
2) Fast, detergent-free samples such as purified proteins or PBS-diluted samples suit Bradford, a five-minute incubation at room temperature, then a read at 595 nm, with a dynamic range of 0.1 to 1.4 mg per ml.
3) Standard BCA assays are susceptible to reducing agents. Diluting your sample at least 1 in 10, a reducing-agent-compatible BCA variant, and a Bradford or Biuret assay all reduce this interference.
4) GFP-tagged protein quantification calls for an anti-GFP sandwich assay kit, detecting GFP in crude lysates without gel separation. It suits work where you need to track the expression level of a GFP fusion construct across conditions.
Applications
Western blot normalisation
Accurate total protein concentration, measured by BCA or Bradford, ensures equal loading across your lanes for reliable band-intensity comparisons.
Assay QC for ELISA and activity assays
Knowing the protein input concentration lets you express activity per mg for ELISA assays, so you can compare runs and spot outliers.
Recombinant protein lot release
Quantification at A280 gives you a rapid, reagent-free concentration check for purified proteins with known extinction coefficients.
Cell lysate standardisation in proteomics
Before mass spectrometry sample preparation, BCA-based quantification of your tryptic digest input keeps peptide loads consistent across samples.
GFP-tagged protein quantification
An anti-GFP sandwich assay detects GFP fusion constructs in crude lysates, tracking expression across conditions without gel separation.
Bulk or high-concentration protein samples
Biuret assays detect peptide bonds in alkaline copper solution, giving you reagent-stable quantification for higher-concentration samples.
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Protein Quantification
Sample-Matched BCA, Bradford & Biuret Protein Quantification Kits
Frequently asked questions
Both BCA and Bradford are sensitive to reducing agents. DTT above about 1 mM causes overestimation in BCA assays by reducing cupric ions directly. TCEP interferes with Bradford. Diluting the sample at least tenfold before assay is often enough. Alternatively, reducing-agent-compatible BCA formulations are available that tolerate higher DTT concentrations.
A280 measurement works best for purified proteins with a known molar extinction coefficient, derived from their tryptophan and tyrosine content. It needs no added reagents and gives results in seconds. It is unsuitable for crude lysates, because nucleic acids also absorb at 280 nm and cause overestimation. The A260/A280 ratio can flag nucleic acid contamination.
Bovine serum albumin (BSA) is the conventional standard. It is inexpensive, stable, and has a well-defined absorbance response. However, BSA can give different readings from your protein of interest, because the colorimetric response depends on amino acid composition. For the most accurate result, use a purified aliquot of the target protein itself as the standard.
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