can a pellet be called "probe"? - (Mar/18/2006 )
homebrew is right.
Except if it'a for checking total proteins before doing an immunopurif or sthg else, the protocol is ok and the "probe" is actually part of your extract.
I think that your protocol means that after the lysis step you have to check if you have well separate your protein from this pellet. And for that your pellet acts as a probe in the fact it allows to answer to check if you have well separate your protein from this pellet
Kathy, would you please attach the reference? Is your protocol from a paper?
It looks as though your protocol is about probing your supernatant for your POI...like perhaps they skipped a step when writing the M&M? I would almost think it was for a western to quantify your protein yield, and that the steps listed are incomplete...because it doesnt' really make sense otherwise...
thank you everyone for your help...
this is the website
i just found it by google search.....the second one (insoluble proteins) says for glutathione purification so i assumed that the first one and last one are for the same purpose...
wow...that doesn't really make sense
the only thing I can think, is that the #1 protocol on that link is pretty poorly written and is very vague about some steps at the end
anyone else have a more clear idea?
Kathy, is it possible to contact the authors of the protocol and ask them to clarify what they were doing?
Or, tell use your experiment, and we'll help you devise a more complete protocol...
aimkins, ill try to contact the authors to seee what they meant....
homebrew, ive expressed my protein in BL21 strain of E-coli and now im trying to lyse the cells and solubilize the protein so i can later on attach it to glutathione sepharose beads....my protein is not soluble i guess.... since all protocol ive tried got me very very little protein in the soluble fraction and most of it in the insoluble fraction....so im trying different protocols right now before going into urea and denaturing stuff.....thanx a lot
Can you give us more detail, Kathy? Like the expression vector you're using, how you determined that your clone was correct, your induction protocol, and a bit about the protein (its size, whether it's a membrane protein, the species it's from, etc.)?
All of these details need to be considered when designing an enrichment protocol.
homebrew, thank you so much for your time, my vector is pGEX-KG and i have inserted my protein ORF by PCR and checked that the insert is there by PCR of the colonies....and i have induced the protein (which should be cytoplasmic) by growing culture to OD 1 then adding 0.5 mM IPTG, then shaking for 6 hours at 31 C.....proteins MW is 91 KDa (with GST) and it is degraded in E-coli so comes out as two bands....(al least two) ....i can see my protein after I boil E coli culture in SDS-loading buffer....but when i try to solubilize my protein into the buffer i fail... i tried lysozyme, sonication different buffers......but up till now no progress..... it just seems so insoluble....its all in the pellets fraction......
did you sequence the vector obtained? i don't think that PCR positive is only sufficient perse. No pcr is ok to tell for a wrong colony but positive...
Btw, i assume it should be ok as you've an induced band. but sequence may tell about missense mutations
may you express your protein it in yeast? did you try to change of strain?
if you are trying to purify an expressed protein from e coli just refer to several manufacturers protocols such as the novogen pET system or the qiagen pQE system. they are very simple and explained ad nauseam