My buffer or components of my buffer are not listed in the compatibility table for my protein assay. What should I do?
You can test the tolerance of the assay for your specific buffer formulation. For in-house generated compatibility information, substances were considered compatible at the indicated concentration in the Standard Test Tube Protocol (found in the manual for each protein assay) if the error in protein concentration estimation caused by the presence of the substance was less than or equal to 10%. The substances were tested using WR prepared immediately before each experiment. Blank-corrected 562nm absorbance measurements (for a 1000µg/mL BSA standard + substance) were compared to the net 562nm measurements of the same standard prepared in 0.9% saline.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
All the components of my sample buffer are at or below the indicated compatible concentration for my protein assay, but I am still seeing too much/too little color development. What could be the problem?
It is possible to have a substance additive affect such that even though a single component is present at a concentration below its listed compatibility, a sample buffer containing a combination of substances could interfere with the assay. You should take steps to eliminate or minimize the effects of the interfering substance(s) by diluting or removing the substance.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
My protein assay is not developing color or is developing too much color. What can I do?
Refer to the information in the product-specific instruction booklet or our Tech Tip: Protein Quantitation Assay Compatibility Table (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
My spectrophotometer doesn’t have a filter set for the absorbance maximum. Can I use an alternate wavelength to read the protein assay?
Often, an alternative wavelength can be used, although the slope of the standard curve and the overall assay sensitivity will most likely be reduced. Our Tech Tip (https://tools.thermofisher.com/content/sfs/brochures/TR0025-Protein-assay-spectra.pdf) offers additional information on determining acceptable wavelengths for measuring protein assays.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
I have cell lysates that were extracted with detergent, which kit should I use for protein quantitation?
The EZQ Protein Quantitation Kit is the most tolerant of non-protein components. It is compatible with samples in SDS-PAGE sample buffer, urea- or guanidine-containing buffers, and detergents. After the protein component is bound to the paper filter, any tracking dyes, detergents, and salts are removed by a methanol wash, so they are no longer present to affect protein quantitation.
Other protein assays that are compatible with detergents are colorimetric BCA assays, Detergent Compatible Bradford Assay Kit, and the 660 nm Protein Assay Kit.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
