Does protein A affect Protein B level in my case? - need advice on my confusing result (Feb/01/2007 )
Hi, everyone:
I am studying whether protein A will affect protein B expression. I transfect His tagged protein A plasmid which is made by using PCDNA3.1 vector, into cells and do simple western to detect protein B level.I use PCDNA3.1 as control. the replicated results showed that protein A dramatically upregulate protein B expression. I am exctied about this. To confirm the result, I transfect no tagged protein in to the cell, still use PCDNA3.1 as control. However, in this case,protein B expression has no change completely.
How this could happen? Which result I should believe in? Is the His tag affect protein B level rather than protein A itself? PCDNA3.1 is not good control?
I am looking for any suggestion and advice.
Thank you very much!!
I dont think the His-tag has much to do with increased expression.
I would suggest to try transfecting cells with protein A ( myc his) and have a non-myc his tagged version and a control (only pcDNA) . So you will have all 3 side by side. Now try the western to detect protien B.
pcDNA is a good choice for control. by the way, western is not really quantitative to compare expression levels, so take care.
Good Luck !!!
I would suggest to try transfecting cells with protein A ( myc his) and have a non-myc his tagged version and a control (only pcDNA) . So you will have all 3 side by side. Now try the western to detect protien B.
pcDNA is a good choice for control. by the way, western is not really quantitative to compare expression levels, so take care.
Good Luck !!!
I do apprecitate your prompt reply. I have done the exp as you said, running the 3 side by side. but still the his tagged version has effect but not the non tagged. As protein B level is very very low in the cell I used, the result showed that in control lane no signal, in exp lane(tansfected with his tagged protein A) there is a big fat band and the size is right. if it was not protein B, what else it could be?
Any further advice?
Thanks again!
I would suggest to try transfecting cells with protein A ( myc his) and have a non-myc his tagged version and a control (only pcDNA) . So you will have all 3 side by side. Now try the western to detect protien B.
pcDNA is a good choice for control. by the way, western is not really quantitative to compare expression levels, so take care.
Good Luck !!!
I do apprecitate your prompt reply. I have done the exp as you said, running the 3 side by side. but still the his tagged version has effect but not the non tagged. As protein B level is very very low in the cell I used, the result showed that in control lane no signal, in exp lane(tansfected with his tagged protein A) there is a big fat band and the size is right. if it was not protein B, what else it could be?
Any further advice?
Thanks again!
you may measure protein A-dependend protein B expression, but one should also show it aon the mRNA level as protein A may downregulate turn-over (proteolysis) of B
I would suggest to try transfecting cells with protein A ( myc his) and have a non-myc his tagged version and a control (only pcDNA) . So you will have all 3 side by side. Now try the western to detect protien B.
pcDNA is a good choice for control. by the way, western is not really quantitative to compare expression levels, so take care.
Good Luck !!!
I do apprecitate your prompt reply. I have done the exp as you said, running the 3 side by side. but still the his tagged version has effect but not the non tagged. As protein B level is very very low in the cell I used, the result showed that in control lane no signal, in exp lane(tansfected with his tagged protein A) there is a big fat band and the size is right. if it was not protein B, what else it could be?
Any further advice?
Thanks again!
I would check the plasmid prep of the non-tagged protein A. I think there might have been a mistake there. Verify it by sequencing or restriction digest.
Also The Bearer's (kosmodrom) suggestion is quite right. You need to know the mRNA levels to be conclusive about the effect of A on B.
Thank you all.
The non tagged version of protein A can express protein A very good(the western shows very strong signal).
I have check the mRNA level. consistent with the protein level, protein B mRNA level also increase!
I may provide more details. the protein B is an improtant adaptor in TNF signaling pathway and TLR4 signaling pathway. Could it be that my plasmid of protein A has been contaminated by LPS so that the TLR4 pathway is activated which can upregulate protein B. The plasmid A is prepared from midi-prep. so anyone has ever met such a problem?
The non tagged version of protein A can express protein A very good(the western shows very strong signal).
I have check the mRNA level. consistent with the protein level, protein B mRNA level also increase!
I may provide more details. the protein B is an improtant adaptor in TNF signaling pathway and TLR4 signaling pathway. Could it be that my plasmid of protein A has been contaminated by LPS so that the TLR4 pathway is activated which can upregulate protein B. The plasmid A is prepared from midi-prep. so anyone has ever met such a problem?
plasmid of protein A should be prepared with an endotoxin-free certified plasmid preparation kit or compared to your preparation protocol if there are differences which may arise from LPS; do it before reviewer ask
The non tagged version of protein A can express protein A very good(the western shows very strong signal).
I have check the mRNA level. consistent with the protein level, protein B mRNA level also increase!
I may provide more details. the protein B is an improtant adaptor in TNF signaling pathway and TLR4 signaling pathway. Could it be that my plasmid of protein A has been contaminated by LPS so that the TLR4 pathway is activated which can upregulate protein B. The plasmid A is prepared from midi-prep. so anyone has ever met such a problem?
plasmid of protein A should be prepared with an endotoxin-free certified plasmid preparation kit or compared to your preparation protocol if there are differences which may arise from LPS; do it before reviewer ask
Thank you very much.
I use a commercial kit to prepare the plasmid(I forget the brand now).
Is any way to test whether my plasmid has been contaminated with LPS?
Someone said take several ul plasmid for digestion to destroy the plasmid structure, then use the digested plasmid to do transfection so that I can tell whether there is LPS contaminated. Do you think it is feasible?Is LPS heat-inactivated during digestion?
What is your suggestion?
Thank you