wrong protein size - (Jul/12/2008 )
Hi. I am new to this protein expression experiments. I am using pCal-n-EK vector to express my protein in BL21 Gold (DE3). In the SDS gel, I found a 38kDa protein induced, However, the size i expecting was 31kda. My gene is in frame at 5' end and it is complete with stop codon (TAG). Please help me, in troubleshooting. What do you think went wrong or .....?
Thanks
It is true that the protein induced is bigger than the protein predicted. Because the protein is modified in E.coli. And sequence the plasmid if you have any problem.
Thanks
that is fairly common. Predicted size is not what you generally get on western, mainly because of post-translational modifications.
A way to check this is to use peptide blocking for your antibody. If the peptide/antigen can block this particular band from appearing in western, you have got a specific band. Then sequence the band by MS/MS to find out what exact modification it is. You will get a figure 3C right there for your paper.
Thanks for your reply. actually i am expressing this protien in E.coli to generate antibody against it. I checked by sequencing. it is correct and in frame.
Regards
Thanks
that is fairly common. Predicted size is not what you generally get on western, mainly because of post-translational modifications.
A way to check this is to use peptide blocking for your antibody. If the peptide/antigen can block this particular band from appearing in western, you have got a specific band. Then sequence the band by MS/MS to find out what exact modification it is. You will get a figure 3C right there for your paper.
Regards
Great! I prefer raising ab against peptide/s. Less involvement, at least in the beginning!
True..... but isn't it expensive. we are trying to save some money. How much it costs thru company.
Regards
Great! I prefer raising ab against peptide/s. Less involvement, at least in the beginning!
Thanks bigreed for your reply. My sequence is correct and in frame. may be some other possibility?