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Author Topic: adsorption control+ unspecific IGG  (Read 2634 times)

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Offline wendy

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adsorption control+ unspecific IGG
« on: May 12, 2006, 07:23:59 AM »
Hey!

I am having some trouble with my preadsorpiton control. Is it a good suggestion to centrifuge the antibody and the blockingpeptide for 20 minutes before usa and only incubate the section with the supernatant? Has anyone tried this way of doing it? I found it recomended on the Chemicon website ...

I also wonder what the difference is with unsepcific IGG and normal serum. Is it ok to incubate negative control sections with normal serum instead of the primary antibody or do you have to use unspecific IgG?

Thank you!

adsorption control+ unspecific IGG
« on: May 12, 2006, 07:23:59 AM »

Offline richard03

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Re: adsorption control+ unspecific IGG
« Reply #1 on: May 19, 2006, 11:33:32 PM »
The use of supernatant can reduce unspecific binding, and it suppose to work better.

I would use IgG isotype matching primary antibody for negative control. It gives you more accurate result. Normal serum may contain several subclasses of IgG.

Offline ImmunoNYC

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Re: adsorption control+ unspecific IGG
« Reply #2 on: May 20, 2006, 01:00:25 AM »
It is my opinion that the preadsorption control really does not mean a whole lot. All it shows is that your antibody binds to whatever peptide or protein was used for immunization. It tells you nothing about any non-specific interactions your antibody may have on your cells or tissue of interest. In my opinion it is an expensive waste of time.

However, that being said a spin down is always a good idea but I cannot understand why one would need to spin for TWENTY minutes? Do you have protein aggregates or particulate matter??? Strange.

Richard's advice to use IgG - preferably isotype matched - rather than serum is a good idea as serum contains not only the Ig similar to your protein but all sorts of other crap and can actually end up giving you more background and make interpretation of your results tricky. Avoid it if possible.
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Re: adsorption control+ unspecific IGG
« Reply #2 on: May 20, 2006, 01:00:25 AM »