Elution from immunoprecipitation suitable for 2D-GE - Elution from immunoprecipitation suitable for 2D gel electrophoresis (Aug/12/2006 )

We have tried to elute immunoprecipitated proteins (utilizing an agarose-immobilized anti-phospho-Tyr Ab) with rehydration buffer (7 M Urea, 2 M Thiourea, 2 % CHAPS) for immobiline strips for 2D-GE. However, rehydration buffer was not fully efficient to elute as there were some proteins left on precipitate shown by re-elution with classical Laemmli-buffer after elution with rehydration buffer. Has someone a simple trick to optimize elution which will not interfere with rehydration of IEF stripes?

We think elution with 0.1 Glycin/HCl pH2.5 is not to recommend. Our only idea is to elute with high Magnesium-salt or commercials such as Immunopure elution buffer, then dialysing against rehydration buffer. However, as we are limited in specific proteins, we might get problems by losing some amount of proteins, and try to circumvent dialysis.

Hi,

We used first dimmension buffer to elute the IP. Yes it is not 100% efficient, but very close. Anyway, even laemmli buffer is not 100% efficient as I have checked.

Regards,

Antonio