problem with antibody blocking - (Feb/07/2010 )

He all!

Help me! I started to do immunolocalization and find out that I have a problem with control samples: cannot get controls with no signal. I use a costom polyclonal antibody raised in rabbit. The immunolocalization works fine and I can see the signal just in the place where it should be but the control samples always looks the same. I have used blocked antibody for my negative controls. The blocking has been obtained with the peptide that the company provided and which was used to raise the antibody. If I leave the antibody out from incubations then I obtain slides with no signal.

I have tested the antibody by western blotting and I can see one band of right size. When I block the antibody with the peptide the band disappears. So the blocking works fine in westerns but not in immunolocalization. I cannot understand why.

Has anyone any idea why this happens? Should I use something else to block the unspecific places (now I have tried BSA and non-fat milk) or block the samples longer time or wash more intensively? Or is there some other explanation that I dont know.

Thank you for replying.

I am trying to detect 20-kDa protein from plant stems embedded in paraffin. I have cut stems 10 um sections. I remove the paraffin with xylene and then do ethanol series up to water. I perform blocking with 5% BSA or 10% non-fat milk in TTBS for 1h at room temperature. I incubate the sections with primary antibody in blocking solution for 2 h. Then wash with TTBS in shaking 4 x 10 min. Then block again for 30 min and incubate in secondary antibody produced in goat against rabbit linked to alkaline phosphatase, 1h in blocking solution. Then wash again and incubate in alkaline buffer and detect with NBT/BCIP. I do incubation (except washing) by adding liquid on slides and covering them with parafilm. Maybe I should put the slides on jars filed with liquid?

Sounds like the 'blocking' is incomplete for the test you are running. By blocking I mean blocking the ab binding sites not NSB blocking. Why not react the primary antibody with antigen/peptide overnight 37C with excess ag?

Another possibility:
Can you use a 'negative-non expression' tissue for your negative control rather than 'blocking' the primary antibody? I think this would be a true negative.

Another option
You could use a completely different ab against another target providing the specices, class, subclass are the same and the protein concentration is the same. This way you don't have to verify the blocking of the binding site of the primary ab.

Thank you for the tips!

I have used similar blocking that was enough to gave one band in western blots. That was 5% BSA or 10% non-fat milk. Maybe I can try saturated milk next and keep in blocking solution for longer periods of time.

I also wonder if "antigen retrieval" is the problem. I have not perform any incubations to sections between xylene removal and blocking. Maybe the antibody can not attach to the antigen. The development of the signal takes usually quite long time: 1 h. Maybe all the colour that finally appears is unspecific. With my collegue the development time is 5 min.

I have done the blocking also with excess of peptide but performed the blocking at +4 degress as it worked for western blottings.

I have no negative non-expression tissue available. I can use only another species for that.

maris on Sun Feb 14 09:27:54 2010 said:

Thank you for the tips!

I have used similar blocking that was enough to gave one band in western blots. That was 5% BSA or 10% non-fat milk. Maybe I can try saturated milk next and keep in blocking solution for longer periods of time.

I also wonder if "antigen retrieval" is the problem. I have not perform any incubations to sections between xylene removal and blocking. Maybe the antibody can not attach to the antigen. The development of the signal takes usually quite long time: 1 h. Maybe all the colour that finally appears is unspecific. With my collegue the development time is 5 min.

I have done the blocking also with excess of peptide but performed the blocking at +4 degress as it worked for western blottings.

I have no negative non-expression tissue available. I can use only another species for that.