The main limitation of the Bradford assay is its incompatibility with most detergents, routinely used to solubilize membrane proteins. (Interestingly, however, very low levels of non-ionic detergent, such as Triton X-100, may improve sensitivity and variability of the Bradford assay [25] ).
Why do detergents interfere with Bradford assay?
Additionally, free amino acids and small peptides (<3 kDa) do not interfere with the assay. If a quick Bradford protein assay kit with a ready-to-use Bradford reagent is used, detergents that interfere will need to be at a lower concentration in the sample than in the standard assay due to the high sample-to-dye ratio.
How does the Bradford protein assay work?
The Bradford protein assay is a time-tested colorimetric assay. When the Bradford reagent (acidified Coomassie Brilliant Blue G-250) binds to proteins, the dye undergoes a color change in the visible spectrum, with the absorbance maximum moving from 470 to 595 nm.
What is the difference between the Bradford assay and BSA?
The Bradford assay is much more sensitive to immunoglobulin G (IgG) than to bovine serum albumin (BSA), so that with IgG the investigator is likely to overestimate the amount of protein in a sample. With BSA the investigator is likely to underestimate the amount. The assay is less accurate for basic or acidic proteins.
How do you measure absorbance with Bradford reagent?
To each tube add 1 ml of Bradford Reagent, and mix gently by vortexing. Using reagent blank to zero the spectrophotometer. Transfer the samples to disposable cuvets and measure the absorbance at 595 nm. Samples can be measured from 2 – 60 minutes after addition of Bradford Reagent.
What is BSA and BGG in Biorad assay?
Bradford Protein Assay Standards Bio-Rad Bradford protein assay kits include either of two different proteins, BSA or BGG, as standards to construct a standard curve for the relative quantitation of the proteins in the samples. For most determinations of protein concentration, relative values are generally sufficient.
