Hey you all,
i am currently trying to express a protein in a GST-vector (pGEX6p1). I already cloned it successfully into the vector and the right frame etc is proven by sequencing as well as control digest. There are no stop codons within the protein or anything.
I did then the protein expression in Bl21 DE3 cells by using two methods: IPTG induction and auto-induction. For both methods i found out, that the protein is made via a dot-blot using anti-GST as well as anti-fusion protein antibody. Purification was done by batch purification with sepharose 4B beads.
Now here is my problem: when i load the elute from the beads on a SDS gel, there is only GST (26kDA band). No protein-GST band (which would be 70kDa). The protein i only find in the supernatant-without GST at its original size (42kDa).
I use lysis buffer with protease stop, so i don't get how this could happen?! Has someone of you had a similar problem? What i already tried was to do 2 purifications in a row, meaning that i incubated the supernatant/unbound fraction from the first round in a second. But even here i only have the band for GST and not the GST-protein.....
Thank you all in advance!