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Overview of protein assays
Protein quantitation is most commonly performed using colorimetric assays. Typical methods for the colorimetric determination of protein concentration in solution include the Coomassie blue G-250 dye-binding assay, the biuret method, the Lowry method, the bicinchoninic acid (BCA) assay, and the colloidal gold protein assay.
The most common protein assay reagents involve either protein-dye binding chemistry (Coomassie/Bradford) or proteincopper chelation chemistry (biuret/BCA).
Every protein assay has limitations depending on the application and the specific protein sample analysed. The most useful features to consider when choosing a protein assay are sensitivity (lower detection limit), compatibility with common substances in samples (e.g., detergents, reducing agents, chaotropic agents, inhibitors, salts, and buffers), standard curve linearity, and protein-to-protein variation.
At Thermo Fisher Scientific, we understand these issues and offer numerous colorimetric and fluorescence assays for detection and quantitation of total protein. Our total protein and peptide quantitation assays are all well-characterized, robust assays that provide consistent, reliable results.
Many techniques have been developed to obtain the best protein yield and purity for different types of cells and tissues, taking into account where appropriate, the subcellular location of the protein and the compatibility of the protein extract with the next step in the experiment.
Summary of substance compatibilities with Thermo Scientific protein assays
n/a = not assayed n/c = not compatible
Note: 660 nm assay is compatible with 5% SDS when used with the optional IDCR component available with the kit.
Refer to individual product instructions for more comprehensive compatibility information
BCA-based assays
- Dilution-free
Eliminates the need for sample and standard curve dilutions, reducing set-up time by up to 80%
- Short assay time
Five-minute, room temperature reaction - Wide assay range
Linear working range for BSA of 20 to 10,000 μg/mL - Uniform
Exhibits similar protein-to-protein variation as traditional BCA Protein Assay and less protein-to-protein variation than dyebinding protein assay methods (Bradford) - Compatible
Unaffected by typical concentrations of most ionic and non-ionic detergents - Colorimetric
Optimal absorbance at 480 nm - Accurate
2X more accurate than Bradford assays
Ordering Information
- Colourimetric
Measure with a standard spectrophotometer or plate reader (562 nm) - Excellent uniformity
Exhibits less protein-to-protein variation than dye-binding methods - Compatible
Unaffected by typical concentrations of most ionic and nonionic detergents - Moderately fast
Much easier and four times faster than the classical Lowry method - High linearity
Linear working range for BSA equals 20 to 2000 μg/mL - Sensitive
Detect down to 5 μg/mL with the enhanced protocol
Ordering Information
- Sensitive
Accurately detect down to 0.5 μg/mL (2 μg/mLin microplate format) - High linearity
Linear working range for BSA equals 0.5 to 20 μg/mL - Colourimetric
Measure with a standard spectrophotometer or plate reader (562 nm) - Compatible
Unaffected by typical concentrations of most ionic and nonionic detergents - Convenient
Microplate and cuvette protocols provided with the instructions - Stable
Kit stable at room temperature; prepared working reagent stable for up to 24 hours
Ordering Information
Coomassie-based assays
- Colourimetric
Measure with a standard spectrophotometer or plate reader (595 nm) - Easy to use
Single reagent; no working reagent preparation required - Fast
Almost immediate colour development; add, mix and read results - Broad range
Detects protein concentration in the range 1 to 1500 μg/mL - Better
Improved linearity and response-uniformity compared to traditional Bradford formulations - Flexible
Microplate and cuvette protocols provided with the instructions and adaptable to several target working ranges
Ordering Information
Protease Inhibitor Cocktails
For Research Use Only. Not for use in diagnostic procedures.