I have to make a decision, I think I need your help with this. I am tyring to get soluble forms of a 30 kD protein expressed. I have used pet17b and pgex4t-1 vectors, expression conditions were 0.4 M IPTG induction, 37 degrees centrigrate, 0,1,2,3,4 hours sampling. FROMthe results, I got various kinds of bands in both sds page and western blots. I could not determine whether my protein is expressed or not but according to the molecular weight of my protein, it is expressed and found in the nonsoluble part as well as fairly seen in soluble part. I have tried one more different condition, 20 degrees and o.1 M IPTG induction but yielded the same results for pgex4t-1 ( did not try for pet17b).
Do you have any idea to get this protein in soluble form and get distinct, clear band appearrance? I have used rabbit to immunize it with a 7aa peptide to get antisera.