HELP! confusing antibody - (Jan/13/2008 )

to Almasy:
hi,thank you very much.yes,i diluted just 1:500 and 1:1000 because i found the backgroud was high as you used more serum.but i think your suggestion is feasible,i do need to do stepwise dilution to identify the ab's titer and i could try to do WB agian with higher serum dilution .
as to FLAG-tagged proteins,yes ,i can detect them with FALG abs-but not always,in fact ,that's another problem i met:sometimes,i couldn't detect the trasiently expressed protein with FLAG abs even though i could promise the transfection efficiency according to the cotransfected GFP plasmid.my protein is a glycosylated membrane protein and i don't know if this has sth to do with the weird situation i encountered:unsure:
anyway,thank you again for your suggestion !
wish you happiness!

hi,
thanks for the explantion,

i guess the following explanation wud be the possible situation with ur antibody,

since u said, peptide raised antibody recognized both GST-protein and peptide in ELISA that mean ur antibody is perfect interms of recognizing epitopes.

the question remains is, y Ab is not detecting the protein from different cell lysates.

possible answer is, as u know bacterial proteins are translationally unmodified, where as ur protein in different cells undergone modification (glycosylation as u mentioned) extensively, whcih cant be detectable with ur peptide antibody.
glycosylated proteins are poorly antigenic. which is y u cant detect the protein....

solution for the problem wud be, to immunize mamalian cell derived whole protein. or look for possible epitopes on the glycosylated protein and immunize those peptides.

gud luk

Hi,
I would suggest that you make sure of the Ab titer. Modification is certainly one of the possible reasons, but we cross our fingers and hope it is not the case. Try all other solutions first.

hi,

when antibody is working with ELISA and WB, titres of Ab are defined to some extent, i agree with u in regard to checking titres with different cell types.

to rule out this possiblity one just need to use higher concentration of Ab over ELISA or WB for cell lysates.

i wud not really invest my time to titrate antibody in this situation...

welcome for comments.