How to release antibodies from beads after IP? - in order to run ELISA after IP, not Western Blot. (Sep/24/2008 )

Hi,

I was wondering if there is any other buffer except SDS-PAGE sample buffer to release antibodies from Protein G or Protein A?

I would prefer to run ELISA rather than Western Blot after IP.

Thanks

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Maybe boil it for 15min and then vortex?

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how can I boil it when there is no buffer? just boil the beads? so antibodies would be released where?...it needs a buffer.

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hi,
no need to boil, once u have washed protein A or G beads, add 10mM glycine (pH-3) incubate for 5 minutes, bring back the pH to 8. use the elute for ELISA.

gud luk

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hi thank you very much, how to bring back to pH8 and how to take down to pH3? just by HCl and NaOH? ordinary chemicals?

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hi,

add conc HCl to bring down the pH to 3
add NaOH to bring back the pH to 8.

before you do the experiment, perform a preliminary experiment to find out the volumes how much to use.
fixed volume of low pH solution
find out how much of NaOH should be added to the fixed low pH solution to bring back the pH to 8.

then proceed with your real experiment.

gud luk

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thanks

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you should make a buffer that is 0.1M glycine, pH 2.5 to elute.

add some 0.5 or 1M tris base (dropwise) to neutralize the eluent (usually 1/10 to 1/20 the fraction volume).

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