Can I read at wavelengths other than 595 nm?
If a photometer or plate reader is not available with a 595 nm filter, the blue color may be measured at any wavelength between 570 nm and 610 nm. The maximum sensitivity of the assay occurs when the absorbance of the Coomassie dye protein complex is measured at 595 nm. Taking the absorbance measurements at any wavelength other than 595 nm will result in a lower slope for the standard curve and may increase the minimum detection level for the protocol.
Can detergents used to clean my glassware interfere with the assay?
Care must be exercised when cleaning glassware that will be used again for protein assays. Thorough cleaning often requires the use of a detergent, it must be completely removed in the final rinse. Also, the Coomassie dye will stain glass or quartz cuvettes. The cuvettes can be cleaned easily with our detergents followed by a thorough final rinsing with deionized water. If you prefer, disposable polystyrene cuvettes may be used to eliminate the cuvette-cleaning chore.
How can I remove interfering substances from my sample before using the Coomassie Plus Reagent?
You can remove interfering substances easily with the use of one of several Pierce products designed to purify your sample. These products include Slide-A-Lyzer Dialysis Cassettes, D-Salt Desalting Columns and Extracti-Gel D Detergent Removing Gel. Furthermore, TCA and cold acetone can be utilized to remove other interfering substances. (See the TechTips section of this web site for more information.)
How does the Coomassie Plus Protein Assay work?
The Coomassie Plus Protein Assay is based on an absorbance shift from 465 nm to 595 nm that occurs when the reagent binds to proteins in an acidic solution. The mechanism of the reaction is the anionic form of the dye complexes with proteins, resulting in a color change from brown to blue. The dye interacts chiefly with arginine residues and weakly with histidine, lysine, tyrosine, tryptophan and phenylalanine residues. The reaction reaches a stable endpoint so absorbance does not shift over time.
Is there any protein-to-protein variation?
All proteins are unique and their varying structures can give variable responses. Each of the commonly used assay methods exhibits some degree of varying color response toward different proteins. These differences relate to dissimilarity among proteins due to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein's color response. Most protein assay methods utilize bovine serum albumin (BSA) or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined. Using either of these proteins as the standard works well in most assay methods. However, if great accuracy is required, the standard curve must be prepared from a pure sample of the target protein to be measured. If a pure sample of the target protein is not available, select the standard protein from those proteins that generate a color response that is close to the color response of the target protein.
What agents are compatible with Coomassie Plus Protein Assay?
The assay is compatible with many agents that are incompatible in other assays. It has been shown to be compatible with amine-containing buffers, reducing agents, chaotropic agents, organic solvents, sugars, antimicrobial agents, DNA, protease inhibitors, and low concentrations of metal chelators and metals.
What are the advantages of the Coomassie Plus Protein Assay over the Bradford Method?
Pierce Coomassie Plus Protein Assay has several advantages over the Bradford Method:
- The standard curve is more linear.
- No reagent preparation is required.
- No dilution or filtering is necessary.
- The reaction is very fast (results in as little as 30 seconds).
What is Coomassie Plus Protein Assay Reagent?
Pierce Coomassie Plus Protein Assay Reagent is a quick and ready-to-use modification of the well-known Bradford, Coomassie dye-binding, colorimetric method for total protein quantitation.1 This Bradford reagent modification greatly reduces the tendency of Coomassie dye-containing reagents to give a characteristically nonlinear response curve. Coomassie Plus Reagent results in a substantially more linear response curve in a defined range of protein concentration. Pierce's special formulation results in significantly less protein-to-protein variation than that which is observed with other Bradford-type Coomassie dye-based formulations. When Coomassie dye binds protein in an acidic medium, an immediate absorbance shift occurs from 465 nm to 595 nm, with a simultaneous color change of the reagent from green/brown (or red/brown) to blue.
What is the detection range of Coomassie Plus Protein Assay?
Pierce Coomassie Plus Protein Assay has a detection range of 1µg/ml to 1,500µg/ml of protein. The protein of interest must have a molecular weight greater than 3,000 daltons to be detected by the assay. For peptides smaller than 3,000 daltons, try using Fluoraldehyde Reagent Solution (Product # 26025), which will fluorescently detect amino groups on small peptide.
What is the effect of temperature on the assay?
The absorbance readings at 595 nm obtained with the Coomassie Plus Reagent are dependent on the temperature of the reagent to some extent. As the reagent temperature increases to room temperature, the 595 nm readings will increase. Therefore, it is important that the Coomassie Plus Reagent be at room temperature during the assay.