You can still use UV absorbance for quantitation, but you must fully denature the protein and eliminate the aggregation by using 6 M (final concentration) guanidine hydrochloride. In fact, this is a general requirement for quantifying protein by UV absorbance using a calculated extinction coefficient and should always be done.

If you don't want to get involved with guanidine HCl, which is hazardous, then use the Bradford assay, Dc Lowry assay or BCA assay. The conditions of these assays are either strongly acidic or moderately basic and should deal with a mild amount of aggregation.

Thank you so much Dr. Adam. I will prepare 12 molar Guandine HCl and take 100 mcL of it and add it to 100 mcL of my protein solution and measure the absorbance and calculate the concentration and multiply it by 2 is that correct?

Thank you it just this is my first time doing protein purification

I don't think guanidine HCl is soluble at 12 M. It is soluble at 8 M and can be purchased at that concentration. If you decide to make the solution yourself, be very careful. Guanidine HCl is harmful to skin and other tissues. Be sure to wear protective personal equipment.

Using 8 M guanidine, you would mix one volume of protein solution with 3 volumes of 8 M guanidine to get to 6 M. The protein concentration would then be multiplied by 4.

Just to add an idea to the conversation,

If the dimerization is not occurring at the dialysis step it is better to quantify your monomeric protein in solution after applying SEC polishing step. One step IMAC purification may cause co-elute oligomeric states of target protein along with monomeric structure besides unwanted residual host cell proteins. whether Elisa or UV-based approach I may prefer two-step tandem purification to ensure absolute quantification...