The Thermo Scientific Pierce 660nm Protein Assay is a ready-to-use, detergent- and reducing agent-compatible assay reagent to quickly measure (A660nm) total protein concentration compared to a protein standard.
The Pierce 660nm Assay is more linear than coomassie-based Bradford assays and compatible with higher concentrations of most detergents, reducing agents and other commonly used reagents. The accessory Ionic Detergent Compatibility Reagent (IDCR) provides for even broader detergent compatibility, making this one of the only protein assays that is suitable for samples containing Laemmli SDS sample buffer with bromophenol blue. Although the Pierce 660nm Protein Assay produces a higher level of protein-to-protein variation (37%) than other assays, such as the BCA Protein Assay, the simpler single-reagent format and broader substance compatibility make the Pierce 660nm Assay more convenient for many routine applications. The Pierce 660nm Protein Assay can be performed in either a test tube or microplate format.
Highlights:
- Versatile – works with a greater range of detergents and reducing agents than other dye-based assays
- Fast – single reagent with a simple mix-and-read protocol; no working reagent to prepare
- Accurate – produces standard curves that are more linear than with the Bradford assay method
- Flexible – assay may be performed in test tubes or microplates
- Conserve samples – requires only 10µL for microplate or 100µL for the test tube procedures
- Convenient – room temperature storage means no waiting for reagent equilibration before use
Product Details:
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Performance comparison of the Bio-Rad Bradford Protein Assay versus the Thermo Scientific Pierce 660nm Protein Assay. Assays were performed according the standard test-tube procedure using 100µL of BSA. The Pierce 660nm Protein Assay has a greater linear range (25 to 2000µg) compared with the Bradford Assay (125 to 1000µg). Absorbances were measured at the appropriate wavelengths for each assay (660nm and 595nm, respectively).
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Typical color response curved using the test tube procedure. The linear detection ranges are 25 to 2000µg/mL for bovine serum albumin (BSA) and 50 to 2000µg/mL for bovine gamma globulin (BGG). Due to the inherent protein to protein variability of all protein assays (37% for the 660nm Protein Assay), this demonstrates that appropriate standards should be used for the type of unknown samples being measured.
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How the Pierce 660nm Assay Detects Protein:
The Pierce 660nm Protein Assay is based on the binding of a proprietary dye-metal complex to protein in acidic conditions that causes a shift in the dye's absorption maximum, which is measured at 660nm. The dye-metal complex is reddish-brown and changes to green upon protein binding. The color change is produced by deprotonation of the dye at low pH facilitated by interactions with positively charged amino acid groups in proteins. Therefore, the dye interacts mainly with basic residues in proteins, such as histidine, arginine and lysine and to a lesser extent tyrosine, tryptophan and phenylalanine.
The color produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations, even in the presence of detergents and reducing agents that would be incompatible with Bradford and BCA Protein Assays. The optional IDCR may be added to the assay reagent to increase compatibility with high amounts of ionic detergents, allowing samples containing Laemmli SDS sample buffer with bromophenol blue to be measured. The IDCR completely dissolves by thorough mixing and does not have any affect on the assay.
For more information, see the article "Chemistry of Protein Assays" in the Protein Methods Library.
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| The absorption maximum of the 660nm Assay Reagent-metal complex shifts proportionally upon binding to BSA. The absorption spectra were recorded for the Pierce 660nm Protein Assay Reagent from 340 to 800nm using a spectrophotometer. Protein in the presence of the reagent-metal complex produces a significant absorbance shift at a wavelength of 660nm. |
References:
- Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-54.
- Gornall, A.G. (1949). Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177: 751-66.
- Lowry, O.H. (1951). Protein measurement with Folin-Phenol reagent. J. Biol. Chem. 193: 265-75.
- Smith, P.K., et al. (1985). Measurement of protein using bicinchonninic acid. Anal. Biochem. 150: 76-85.
- Stoscheck, C.M. (1987). Protein assay sensitive at nanogram levels. Anal. Biochem. 160: 301-5.
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