23225-BCA-Assay-Kit-230

In 1985, Paul K. Smith, et al. introduced the BCA Protein Assay. Since then it has become a popular method for colorimetric detection and quantitation of total protein. The BCA Protein Assay has a unique advantage over the Modified Lowry Protein Assay and any of the Coomassie dye–based assays—it is compatible with samples that contain up to 5% surfactants (detergents). Compared to most dye-binding methods, the BCA Assay is affected much less by protein compositional differences, providing greater protein-to-protein uniformity.

Briefly, the sample is added to the tube or plate containing the prepared BCA Working Reagent and after a 30-minute incubation at 37°C and cooling to room temperature, the resultant purple color is measured at 562 nm. The protocol is similar for the Micro BCA Protein Assay, except the ratio of sample volume to working reagent is different and the tubes are incubated for 60 minutes at 60°C.

Step 1 Step 2 Step 3 Step 4 Notes
Combine 50 parts “A”
and 1 part “B”, mix to make working reagent
Combine 50 μL sample + 1 mL working reagent, mix well Incubate 30 min at 37°C, then cool Read at 562 nm
  • Total assay time:
    100 minutes
  • Requires heating
    for color development
  • Simple, easy to perform
  • Flexible incubation
    protocol allows
    customization of reagent sensitivity and working range
Step 1 Step 2 Step 3 Step 4 Notes
Combine 25 μL
sample + 25 μL
Compatibility Reagent,
incubate 15 min at 37°C
Add 1 mL BCA
working reagent,
incubate 30 min at 37°C
Cool samples 5–10 min at room temperature Read at 562 nm
  • Total assay time: 115 minutes
  • Requires heating for color development
  • No precipitation step
    means no worries about
    difficult-to-solubilize proteins
Step 1 Step 2 Step 3 Step 4 Notes
Combine 50 parts “MA”
+ 48 parts “MB”
+ 2 parts “MC”, mix to make working reagent
Combine 1.0 mL sample + 1.0 mL working reagent, mix well Incubate 60 min at 60°C, then cool Read at 562 nm
  • Total assay time: 130 minutes
  • 60°C water bath is needed
  • Simple, easy to perform

Assay overviews

  • BCA Protein Assay—green-to-purple (A562 nm), two-component, precise, detergent-compatible assay reagent to measure total protein concentration vs. protein standard 
  • BCA Protein Assay - Reducing Agent Compatible—reducing agent–compatible version of our popular BCA Reagent Kit to measure protein concentration in samples containing DTT or BME thiol-reductants. 
  • Micro BCA Protein Assay—special 3-component version of our popular BCA Reagent Kit to measure total protein concentration of dilute protein solutions (0.5 to 20 μg/mL). 

Applications

  • Determine the amount of IgG coated on plates
  • Measure the amount of protein covalently bound to affinity supports
  • Measure column fractions after affinity chromatography
  • Estimate percent recovery of membrane proteins from cell extracts
  • High-throughput screening of fusion proteins
  • Study protein:protein interactions
 
  BCA Protein Assay
1 L/kit
BCA Protein Assay
500 mL/kit
BCA Protein Assay - Reducing Agent Compatible Micro BCA Protein Assay
Applications Determine the amount of IgG coated on plates
Measure the amount of protein covalently bound to affinity supports
Determine copper levels using a reagent formulated with BCA Reagent A
Determine the amount of IgG coated on plates
Measure the amount of protein covalently bound to affinity supports
Determine copper levels using a reagent formulated with BCA Reagent A
Allows the use of the
superior BCA Assay in
situations in which it is
normally unable to be read
Suitable for determining protein concentration in very dilute aqueous solution
Uniformity Less protein:protein variation than Coomassie dye methods Less protein:protein variation than Coomassie dye methods Significantly less (14–23%) protein:protein variation than Bradford-based methods Less protein:protein variation than BCA, Coomassie dye, or Lowry methods
Works with peptides Yes, three amino acids or larger Yes, three amino acids or larger No Yes, three amino acids or larger
Cuvette assay range 20 to 2,000 (50 µL)
5 to 250 (50 µL)
20 to 2,000 (50 µL)
5 to 250 (50 µL)
125 to 2,000 (25 µL) 0.5 to 20 (500 µL)
Microplate assay range 20 to 2,000 (25 µL) 20 to 2,000 (25 µL) 125 to 2,000 (9 µL) 2 to 40 (150 µL)
Stability of working solution One week at RT One week at RT 8 hours at 4°C protected from light 24 hours at RT
Compatibilities Detergents Detergents Detergents
Reducing agents
Detergents
Incompatibilities Reducing agents; chelators Reducing agents; chelators Chelators Reducing agents; chelators
Product size 1 L/kit 500 mL/kit 275 mL/kit 500 mL/kit
Order Now Order Now Order Now Order Now

We offer easy-to-use colorimetric or fluorescent peptide assays that are designed specifically to improve the sensitivity and reproducibility of quantitation of peptide mixtures. The kits include simple protocols with a representative standard enclosed. The kits have been fully validated with representative samples that have been processed and analyzed using Thermo Scientific™ Mass Spectrometers