high background with peptide blocking - (Apr/13/2007 )

I am characterizing a new antibody on mouse brain sections. The antibody gives a good signal. To show the specificity of the antibody, I did a blocking experiment with the peptide. Briefly,I preincubated 1 microgram of the antibody with 1 microgram of the peptide (used to make the antibody) (which is 100x in terms of molarity) in 100 microL of PBS at 4°C for 90 min. I then centrifuged the mixture for 5 min at 13,000 g, and put 90 microL from that into the blocking solution over sections o/n at 4°C. I used DAB reaction for staining.

The problem is that the background increases considerably with antibody+peptide, as compared to when antibody is used alone. Can someone guide me how to reduce this background.

Thanks

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I remember experiments with similar experiences; since that time I pass on blocking peptides in immunoblotting

In my case the blocking peptide bound to some blotted proteins in the way of a far Western blot; the peptide was of course detected by the Ab; so you get a ternary complex of peptide-binding protein/peptide/Ab;

you need an alternative peptide or the whole protein for Ab blockade, and the hope that these alternatives do not bind to Western blotted proteins; I think any protocol to lower affinity of peptide and peptide-binding proteins may affect the Ab-antigen binding

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Did you try incubating the peptide-antibody mixture overnight or at least longer than 90 min?

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in my experience longer incubation of peptide with Ab did not help as, in my case, the peptide binding site to the blotted protein was not masked by the Ab; so, I think the peptide/Ab complex still bound with the peptide; nevertheless, in the case of blocking the peptide binding site with a binding-site masking Ab should circumvent the problem...

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Thanks for your replies.

"blocking the peptide binding site with a binding-site masking Ab should circumvent the problem" How can I know where the peptide binding site on other proteins lies?

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by expressing and blotting various deletion forms of your protein, or more simple, separating tryptic peptides of your protein, blotting and reprobing; if there is enough protein it may be enough for Ms/Ms analysis or Edman sequencing

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