A great case problem on antibody assay - (Feb/11/2007 )
Could anyone give me a reasonable answer on my problem?
I have picked gel spots from 2D gel and immunize the rat. However, the result from western blotting shows that the band (which the antibodies against the protein ) has shifted for a few molecular size. The specificty is so strong that it is not a non-specific binding.
-HenryTcby-
QUOTE (HenryTcby @ Feb 11 2007, 05:22 PM)
Could anyone give me a reasonable answer on my problem?
I have picked gel spots from 2D gel and immunize the rat. However, the result from western blotting shows that the band (which the antibodies against the protein ) has shifted for a few molecular size. The specificty is so strong that it is not a non-specific binding.
I have picked gel spots from 2D gel and immunize the rat. However, the result from western blotting shows that the band (which the antibodies against the protein ) has shifted for a few molecular size. The specificty is so strong that it is not a non-specific binding.
how many rats did you immunize? (there is much luck to get with 1 immunization a good working Ab)
how was the Ab tested to be specific?
As I think you test it specificity, I suggest the following posssibilities:
if the Ab is specific against your protein of interest, the antigen shift may explained by by PTM (acylations, glycosylations);
or your polypeptide form 2D gel was a product of degradation; do you know the protein and can deduce molecular mass from primary sequence?
-The Bearer-
QUOTE (The Bearer @ Feb 12 2007, 03:46 AM)
QUOTE (HenryTcby @ Feb 11 2007, 05:22 PM)
Could anyone give me a reasonable answer on my problem?
I have picked gel spots from 2D gel and immunize the rat. However, the result from western blotting shows that the band (which the antibodies against the protein ) has shifted for a few molecular size. The specificty is so strong that it is not a non-specific binding.
I have picked gel spots from 2D gel and immunize the rat. However, the result from western blotting shows that the band (which the antibodies against the protein ) has shifted for a few molecular size. The specificty is so strong that it is not a non-specific binding.
how many rats did you immunize? (there is much luck to get with 1 immunization a good working Ab)
how was the Ab tested to be specific?
As I think you test it specificity, I suggest the following posssibilities:
if the Ab is specific against your protein of interest, the antigen shift may explained by by PTM (acylations, glycosylations);
or your polypeptide form 2D gel was a product of degradation; do you know the protein and can deduce molecular mass from primary sequence?
thank you for your explanation.
I am not sure why the molecular weight of the target proteins have been changed. The protein gel spots were picked from 2D gel. In the case of the western blotting, I choose 1D gel electrophoresis.
The protein should be a novel one.
And there are isoforms of that target protein that shows the same molecular weight as the case of 2D gel (Other antibodies).
-HenryTcby-