how to prepare 2d sample after coimmunoprecipitation - (May/25/2008 )
Hi,everybody.
my experiment is to investigate the proteins interacting with EGFR after some stimulations. So i do co-immunoprecipitation with an anti-EGFR antibody ,get the interacting proteins and identify them. However I meet a lot of difficulties since no one in our lab has run 2D gel after co-immunoprecipitation.
the first problem we have to solve is which buffer we can choose to reprecipitate the immuno-complex. we use SDS sample buffer to reprecipitate the immuno-complex ,boil for 5min, spin the beats and load up. But now for 2D,we can't use this sample buffer. the sample buffer in our lab for 2d have 8 M urea, 4% CHAPS, 40 mM Tris base, 1% DTT，Is that strong for repreciptating the immuno-complex? is it necessary to boil the sample？someone tell me boiling is forbidden for buffer containing urea, for its influence on protein. so what should I do?
I used just the rehydration buffer for isofocusing to wash the beads and get target protins, then this can be directly load to first dimension. Though a little more stricks will appear on the entire gel images, the final result is acceptable.
my experiment is to investigate the proteins interacting with EGFR after some stimulations. So i do co-immunoprecipitation with an anti-EGFR antibody ,get the interacting proteins and identify them. However I meet a lot of difficulties since no one in our lab has run 2D gel after co-immunoprecipitation.
the first problem we have to solve is which buffer we can choose to reprecipitate the immuno-complex. we use SDS sample buffer to reprecipitate the immuno-complex ,boil for 5min, spin the beats and load up. But now for 2D,we can't use this sample buffer. the sample buffer in our lab for 2d have 8 M urea, 4% CHAPS, 40 mM Tris base, 1% DTT，Is that strong for repreciptating the immuno-complex? is it necessary to boil the sample？someone tell me boiling is forbidden for buffer containing urea, for its influence on protein. so what should I do?
my experiment is to investigate the proteins interacting with EGFR after some stimulations. So i do co-immunoprecipitation with an anti-EGFR antibody ,get the interacting proteins and identify them. However I meet a lot of difficulties since no one in our lab has run 2D gel after co-immunoprecipitation.
the first problem we have to solve is which buffer we can choose to reprecipitate the immuno-complex. we use SDS sample buffer to reprecipitate the immuno-complex ,boil for 5min, spin the beats and load up. But now for 2D,we can't use this sample buffer. the sample buffer in our lab for 2d have 8 M urea, 4% CHAPS, 40 mM Tris base, 1% DTT，Is that strong for repreciptating the immuno-complex? is it necessary to boil the sample？someone tell me boiling is forbidden for buffer containing urea, for its influence on protein. so what should I do?
it's really a great help. thank you very much, lavenderyy.
now i have another question, need i boil my sample after resuspention in the rehydration buffer ,or just put it in room temperature for minutes and load it for isofocusing ?
I would not wash the beads in rehydration buffer because the urea and CHAPS will wash your interactors off. Wash in your IP buffer and then elute in rehydration (sample) buffer. Do not boil urea, it will carbamoylate the protein (makes a train of spots on 2D). Leave it at RT for 30 min should be sufficient to elute.
However, I have a suggestion. If you are expecting membrane or large (100 kD +) proteins, do not use 2D gels. These proteins are not well-resolved on 2D. You'll be better off running 1D SDS-PAGE and then compare the control IP to your EGFR IP to get the specific bands. Run a grandient gel if it helps, but cut out bands from both lanes for mass spec. And even better if you have another way of eluting your IP without boiling in sample buffer (eg. elution with peptide), because most of the background binders stick to beads and boiling elutes them all.