I have purified an IgG from cell supernatant using a Protein G column.
Following this size exclusion chromatography was used to determine aggregates of the IgG, monomeric antibody and antibody fragments.
Fractions from SEC were then analysed in a calorimetric assay using Bradford reagent.
As the dye in Bradford's reagent binds to positive amino acid residues such as lysine and arginine, would protein aggregation effect the binding as less amino acids in the IgG will be exposed as some they would be buried within the core of the aggregation?
Of course the ratio of protein:dye will be affected. The real question is does it really matter? Bradford's method is semi-quantitative to begin with (unless you are quantifying a pure single protein using the same protein as standard).
The Bradford reagent contains a high concentration of phosphoric acid. This should disrupt any protein structure or aggregate. For accurate measurements of IgG concentration, use IgG as the standard, as suggested by Omar Gonzalez-Ortega.
Rachael - It's a great question. I have seen scientists asking similar questions about protein aggregates potentially impacting assay performance and data analysis. For your IgG fractions, assuming they are clearly resolved, you can determine IgG concentration (and IgG in aggregate fraction) more accurately by A280 method. Theoretical extinction coefficient using Nick Pace's (1995) equation works very well for most IgGs. So all you need is the sequence. And then measure UV at 280 nm of a dilute solution (A280
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