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Preabsorption of Ab with Ag still leads to staining in ICC


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#1 mastcells

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Posted 04 August 2009 - 05:46 PM

So I've been doing ICC on some mast cells to detect the presence of a protein. The normal procedure led to staining. Leaving out the primary antibody resulted in no staining, therefore we know that the secondary antibody is specific to the primary antibody. Now when we pre-absorbed the antibody with the antigen, we still got staining. Does this mean that either the antigen didn't block my antibodies, the exogenous antigen was displaced by the endogenous antigen, or its binding to some other protein? Because as of right now, I can't say authoritatively say that there is or isn't any of my protein of interest in my cell, because if its binding to something that leads to staining, for all i know it could still be my protein. Any suggestions for how i can figure out what wrong?

#2 HomeBrew

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Posted 04 August 2009 - 06:08 PM

There are a lot of possibilities here... How was your primary antibody prepared? If it's a polyclonal mix, perhaps some of the antibodies don't recognize your blocking antigen because your blocking antigen isn't in the same three dimensional form it was in when the antibodies were produced against it (although I'm not sure how these unadsorbed antibodies would then recognize your target protein when immobilized on a membrane...)..

Or, you haven't used enough blocking antigen to completely block your primary antibody.

What did you run on your gel and transfer to your membrane? Was it a whole cell lysate? If so, was the band that lit up after treatment with secondary antibody the right size? Was there only one band?

Give us some more details on your procedure and the antibodies you're using, and we can be of more help...

#3 mastcells

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Posted 04 August 2009 - 06:59 PM

There are a lot of possibilities here... How was your primary antibody prepared? If it's a polyclonal mix, perhaps some of the antibodies don't recognize your blocking antigen because your blocking antigen isn't in the same three dimensional form it was in when the antibodies were produced against it (although I'm not sure how these unadsorbed antibodies would then recognize your target protein when immobilized on a membrane...)..

Or, you haven't used enough blocking antigen to completely block your primary antibody.

What did you run on your gel and transfer to your membrane? Was it a whole cell lysate? If so, was the band that lit up after treatment with secondary antibody the right size? Was there only one band?

Give us some more details on your procedure and the antibodies you're using, and we can be of more help...


If I wasn't clear, I'm doing ImmunoCytoChemistry not Western Blotting. So I'm fixing my cells onto slides, blocking non-specific binding sites with a blocking serum or dehydrated milk fat, then incubating with either antibody solution or Ab/Ag complex (that has incubated overnight at 4C). Allow primary antibody or Ab/Ag complex to incubate overnight at 4C. Wash. Incubate for 30min with secondary antibody. Wash. and then go through with my staining procedure.

#4 HomeBrew

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Posted 04 August 2009 - 08:23 PM

If I wasn't clear, I'm doing ImmunoCytoChemistry not Western Blotting.


Sorry -- you were clear. I had just read your other post discussing western blotting and had that in my head when replying here...

So I'm fixing my cells onto slides, blocking non-specific binding sites with a blocking serum or dehydrated milk fat, then incubating with either antibody solution or Ab/Ag complex (that has incubated overnight at 4C). Allow primary antibody or Ab/Ag complex to incubate overnight at 4C. Wash. Incubate for 30min with secondary antibody. Wash. and then go through with my staining procedure.


I've never done ICC, but some of my questons are still pertinent (I hope). Is your primary antibody monoclonal or polyclonal? How do you know there's enough Ag in your Ab/Ag complex to block all the Abs?

Additionally, why are you incubating the primary antibody or Ab/Ag complex overnight? Perhaps a shorter time would produce more specificity?

#5 mastcells

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Posted 05 August 2009 - 05:52 AM

I've never done ICC, but some of my questons are still pertinent (I hope). Is your primary antibody monoclonal or polyclonal? How do you know there's enough Ag in your Ab/Ag complex to block all the Abs?

Additionally, why are you incubating the primary antibody or Ab/Ag complex overnight? Perhaps a shorter time would produce more specificity?


The antibodies are polyclonal. And we incubate them overnight per the protocol that came with our ABC staining kit from Santa Cruz Biotech. It said either incubate at room temp for 3 hours or overnight at 4C. I guess I could try doing it for only three hours.

As for how do i know if im adding enough antigen. In my incubation tube, I add 200 nanograms's worth of antibody with 25 micrograms of antigen. Considering that the IgG is about 150 kDa and the antigen is 16kDa, by my rough calculation I have approx. 1000x more antigen than anibody. I could be wrong, but I think that should still be plenty, no?

#6 mastcells

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Posted 05 August 2009 - 08:13 AM

Thanks for your help HomeBrew, but I think we found the source of our problems. When we purchased our antigen that we were going to use to block our antibody, the company made no note on their website that it was not the whole protein. Turns out its 146 amino acids of the 167 (missing the first 20 aa from the amino end and the last amino acid from the carboxy end). Our polyclonal antibodies were supposed to bind epitopes from 22-167. You can probably see where the problem is. If some of the antibody clones can't bind to the protein because that last amino acid doesn't exist on the Ag, then obviously they will be free bind to proteins later on and to produce staining. I'll try and see if I can raise some hell with the company who we bought the protein from for not mentioning that they had cleaved off the last amino acid without making any mention of it on their website. The only place they put this info was on the packing slip once the product arrived.

#7 HomeBrew

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Posted 05 August 2009 - 10:22 AM

Sorry you got bad news, mastcells -- but at least you figured it out...

I suspected there was an Ab/Ag mismatch somewhere -- either in amounts or epitopes....

#8 cellcounter

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Posted 05 August 2009 - 12:13 PM

Thanks for your help HomeBrew, but I think we found the source of our problems. When we purchased our antigen that we were going to use to block our antibody, the company made no note on their website that it was not the whole protein. Turns out its 146 amino acids of the 167 (missing the first 20 aa from the amino end and the last amino acid from the carboxy end). Our polyclonal antibodies were supposed to bind epitopes from 22-167. You can probably see where the problem is. If some of the antibody clones can't bind to the protein because that last amino acid doesn't exist on the Ag, then obviously they will be free bind to proteins later on and to produce staining. I'll try and see if I can raise some hell with the company who we bought the protein from for not mentioning that they had cleaved off the last amino acid without making any mention of it on their website. The only place they put this info was on the packing slip once the product arrived.

This makes perfect sense, but I would probably not raise hell with the company yet. When I do a blocking expt, I always make sure, even if that involves talking to a company representative in Japan, that the protein or peptide that I am purchasing is exactly what was used for antibody generation.

While at it, I would also suggest that even if you had/get an identical antigen, conditions for pre-adsorption and conditions for ICC may vary, and what clones may bind in ICC expt, may not have bound in pre-adsorption expt because of differences in buffer, pH, salts, detergents, denaturation levels, temperature etc. In essence, pre-adsorption may not give you 100% blocking all the time.




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