I have periplasmic protein and it's 31 kDa.
I applied the Bradford procedure to calculate the elution amounts of the proteins after purification. Then I did Dialysis and then bradford again. However, the results are inconsistent. Dialysis membrane 14 MWCO. After dialysis, I put the sample in the Lyophilizer device. When I weigh the dry amount remaining after the lyophilizer, it does not match the dialysis result. I lost about 20 milligrams of protein. Can you help me not to lose proteins? Thanks
It would be helpful if you would provide more information on the protein quantities and concentration measurements.
After purification, I measured with Bradford at 595 nm and found 10 mg/ml in 1 ml of elution buffer (I added the Bradford solution after diluting the proteins). There was 28 mg of protein in a total of 3 elutions (3 ml). 8 mg/ml protein was found when I measured it with Bradford solution at 595 nm after dialysis. The amount of solid after the lyophilizer was about 4 mg.
It is unusual to lose a large proportion of the protein during dialysis, unless (1) it precipitates, (2) the protein is small enough to pass through the dialysis membrane, (3) the protein suffers proteolysis during dialysis and the resulting peptides are lost through the membrane, (4) the membrane has a hole in it or is not properly sealed. Another possibility is that dialysis may have removed one or more substances that were causing interference in the Bradford assay, resulting in an overestimate of the amount of protein.
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