HELP! confusing antibody - (Jan/13/2008 )
hello,everyone!I have a question about the antibody we produced in our lab.
I uesd either GST fusion protein purified from bacteria or peptide synthesised by the company as the antigen to immuninize rabbit and get the rabbit serum.In order to test the efficiency of the antibody,we did ELISA.The reaction was good and seemed workable,but what's confused me is that when I use the serum or purified serum to do western blot,it couldn't detect the endogeneous protein in cells. 
I tried many times and the result were bad.I wondered if someone had met this situation,was there any problem with my antibody?ANY suggestion will be appreciated !
hi,
which serum did u use for WB, is it GST-protein or peptide immunized?
or did u immunize the animal with a mixture of both protein and peptide?
sravan.
I uesd either GST fusion protein purified from bacteria or peptide synthesised by the company as the antigen to immuninize rabbit and get the rabbit serum.In order to test the efficiency of the antibody,we did ELISA.The reaction was good and seemed workable,but what's confused me is that when I use the serum or purified serum to do western blot,it couldn't detect the endogeneous protein in cells.
I tried many times and the result were bad.I wondered if someone had met this situation,was there any problem with my antibody?ANY suggestion will be appreciated !which serum did u use for WB, is it GST-protein or peptide immunized?
or did u immunize the animal with a mixture of both protein and peptide?
sravan.
I uesd either GST fusion protein purified from bacteria or peptide synthesised by the company as the antigen to immuninize rabbit and get the rabbit serum.In order to test the efficiency of the antibody,we did ELISA.The reaction was good and seemed workable,but what's confused me is that when I use the serum or purified serum to do western blot,it couldn't detect the endogeneous protein in cells.
I tried many times and the result were bad.I wondered if someone had met this situation,was there any problem with my antibody?ANY suggestion will be appreciated !hi,thank u for your reply.
I immunized the animal with protein and peptide separately and have done WB with both kinds of sera,they detected the purified protein run on the gel,but couldn't detect the target protein in cells.Do u have any suggestion?thank you very much!
you are sure that your protein expresses in the cell line that you are testing? Is it abundant, or at least with high enough amount to be detected? Is that cell line from the same species with the antigen you used for making Ab? How is the antibody's titer? When you do WB with the fusion protein, how little protein can be detected? At what dilution of Ab? You may want to try several cell types or different WB conditions, depends on the type of protein you are checking. It is also possible that the endogenous protein got modified, so the Ab cannot recognize the epitope anymore. But check the other possibilities first.
It's possible that you produced anti-GST antibodies in fact
hi,
so ur antibodies works in ELISA and WB with recombinant protein,
but they fail to recognize target protein in cells???
if my second statement is right then u shud consider the comments of ALMASY about protein conc in the cells..
according MISSELE there is a posibility of raising antibodies against GST but that is possible only with GST-protein but not with the peptide immunized serum.
regards
hi,thank u for your reply.
I immunized the animal with protein and peptide separately and have done WB with both kinds of sera,they detected the purified protein run on the gel,but couldn't detect the target protein in cells.Do u have any suggestion?thank you very much!
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thank u all for suggestions 
yes,there is possibility that most of the antibodies are generated to GST and that's the reason I used peptide linked to KLH the next time.what's more,when i did ELISA,I used the peptide and the GST fusion protein as the substrate separately and both reactions are good,so we can exclude this possibility as to the antibody generated by the pepetide,right?
as to the expression level,i indeed tried many kinds of cell lines,it's weird that the when dilutied 1:1000 or 1:500,the serum could't detect the exogenous FLAG tagged protein which was transciently trasfected in 293t cell lines,that's a situation the protein was overexpressed,right?
maybe it's the last possibility that the protein was modified,how can i prove that?
hi,
i wud suggest u to be more specific when u ask a question..
since u combine ELISA and WB; peptide and protein u need to explain clearly to get a sensible answer as several combinations and permutations are possible which need different way of analysis.
anyway, when u say modified protein....
u mean the protein in the cell lysate is modified hence the peptide Ab and protein Ab can't recognize the cellular protein levels?
i hope u will be patient and understand the question.
i wish u gud luk
You only use dilutiion 1:1000 and 1:500? If so, I would suggest that you try a bit more. Depend on the Ab titer, you may need to use more Ab for it to work. That is why I ask about the titer and whether you did WB with GST-fusion protein. If you could run a gel with different amount of purified GST-protein, and GST as control, and see how little protein could be detected by your Ab. For this, I would suggest use as little as 10ng to about 5ug. Good Ab should be able to detect the ng protein.
As for FLAG-tagged protein, you can detect the protein using FLAG Ab, I assume?
to donot lie for ever:
hi,thank you for your suggestion,maybe the situation with my ab is too complex or my english is not good enough to elucidate the problem,i'll try to expatiate on the problem again :
At first, i used GST protein as antigen to genenrate antibodys,when i applied the serum to do WB,it couldn't detect the protein transiently expressed in 293t cells or the endogenous protein in many kinds of cell lines.but it did detected the GST protein(the antigen) run on the gel.
i considered the possibility that most of the abs were specific to GST,so we used the KLH linked peptide as antigen to immune animal the next time and in order to test the efficiency of this serum,we did ELISA.as to ELISA,we used the peptide and the GST protein as substrate seperately ,this serum genenrated with peptide reacted well with both kinds of substrate.i think the ELISA results can indicate:1 this serum could detect the antigen efficiently 2 the serum were not specific to GST OR KLH
my lab donot specialise in producing abs and my aim is just to get the ab that can detect my protein in WB,or IHC experiment,the better.my problem is that even this serum genenrated with peptide could't detect the transiently expressed or the endogenous proteins in WB .i don't know if there was sth i missed or if any method could be improved?
best regards