Problems detecting my band!
Posted 09 December 2009 - 08:02 PM
I'm trying to do in vitro expression for some genes. I'n new in it and I'm using the TNT sp6 Wheat germ to clone my genes and then use that cell-free expression system. I cloned my genes with a region in the pF3K vector to use Anti-HisG-HRP Antibody. All of them were then transformed into E.coli DH5a except one, that I needed to clone into the E.coli BL21 (DE3) pLYSs. After performing the expression 2h at 25 °C from the plasmid, I performed the western blotting. All of my clones had the correct band, except that one that I had to cloned into E.coli BL21 (DE3) pLYSs in which I couldn't see any band.
The sequence is OK, so I'm not sure why I can't see anything... Could anyone help me?
Posted 10 December 2009 - 05:53 AM
Posted 13 December 2009 - 01:02 AM
I assume you induced all the cultures with IPTG? What concentration did you use? Could you have failed to induce the pLYSs culture?
In my case, I don't need to express in E.coli, it's in vitro expression with the TNT SP6 High-Yield Wheat Germ Protein Expression System (cell free protein expression). What I was trying to say is that the only difference in my protocol is the E.coli strain in which I cloned my plasmids. In this case, you do de in vitro transcription only by means of the isolated plasmid. I'm not sure if the isolation of the plasmid from that strain can cause any disadvantage for the subsequent in vitro translation.