How to confirm the band of interest in Westerns blot - (Aug/05/2013 )

I am studying a novel protein and we have a probable mwt based on the amino acid seq. However, we have no clue about its glycosylation or phosphorylation level. Neither we know much about its functional role except that its coregulated and coexpressed with TFPI and seems to be playing a role in thrombosis. 

Now, we generated 2 primary antibody for it and am getting 2-3 bands in different tissue extracts i.e I have covered almost all tissues/organs (some has 2 some has 3).

I am puzzled up whether all these 2-3 bands are different variants of proteins as its often seen or non-specificity of antibody (we asked a company to make it for us and its affinity purified.....seems to work well in immunostaining)  

Question: How can i make sure that the band am looking is the same of my protein?

Is there a way that i remove the glycosylation (if there might be any) and the extra bands might go away in some tissue extracts? 

The easiest way to do this is to use the peptide the antibody was raised against - run a gel with some samples you expect to be positive and negative in duplicate groups so that the gel would have something like this: Ladder, +ve, -ve, Ladder, +ve, -ve. 

Transfer the gel onto a membrane and cut the membrane into two so that you now have the two groups of samples on separate sheets.  Probe one half as you would normally with the antibody, probe the other half with antibody that has been pre-incubated with a 5-10 fold excess of the peptide the antibody was raised against.  The specific band should disappear.

Note that the "specific" staining in your immuno could well be these bands that you see in your western, and also that westerns are typically denaturing, whereas immuno usually detects the native form(s) of the proteins.  Not all antibodies will work for both!

You can also do RNAi to knock down the protein and run western to see which band(s) decreases or disappear.

attack it by using siRNA for gene of interest  OR siRNA or INHIBITOR of upstream tareget.

bob1 on Mon Aug 5 20:46:53 2013 said:

The easiest way to do this is to use the peptide the antibody was raised against - run a gel with some samples you expect to be positive and negative in duplicate groups so that the gel would have something like this: Ladder, +ve, -ve, Ladder, +ve, -ve. 

 

Transfer the gel onto a membrane and cut the membrane into two so that you now have the two groups of samples on separate sheets.  Probe one half as you would normally with the antibody, probe the other half with antibody that has been pre-incubated with a 5-10 fold excess of the peptide the antibody was raised against.  The specific band should disappear.

 

Note that the "specific" staining in your immuno could well be these bands that you see in your western, and also that westerns are typically denaturing, whereas immuno usually detects the native form(s) of the proteins.  Not all antibodies will work for both!

Thanks a lot...  

memari on Tue Aug 6 03:01:37 2013 said:

attack it by using siRNA for gene of interest  OR siRNA or INHIBITOR of upstream tareget.

Thanks :)

pcrman on Mon Aug 5 21:07:59 2013 said:

You can also do RNAi to knock down the protein and run western to see which band(s) decreases or disappear.

Thanks :)