I'm running 2D SDS-PAGE after immunoprecipitation. I'm eluting the protein complex with IPG strip rehydration buffer. However I feel I'm not able to complelely elute the complex from bead. Any suggestions on how to optimize the elution before I proceed for 2D? Thanks in advance.
Hi Kamalika,
could you please give a description of your 2D rehydration buffer. Only to check if you could make it more stringent for your elution.
Thank you Eve and Murat. This is the usual 2D strip rehydration buffer fromBioRad with Urea, CHAPS and carrier ampholytes. I've incubated the protein G dynabeads with this buffer for 1h upto 3h; but still failing to elute completely, I suppose.
The urea concentration of the Bio Rad 2D rehydration buffer is 8M so there is not much space left for increasing the urea concentration. And I would also not recommend to boil the sample as long as the eluate will contain urea. This may lead to an undesired carbamylation of the proteins of interest.
Kamalika, how could you know that your interaction partners are not eluted? Did you checkt the elution with a western blot for your bait? Or by identification of interaction partners by LC-MS?
The LC-MS might be the best way to go if your proteins of interest are not solubilizing in 2D sample buffer.
Best,
Murat
Hi Eve,
Sorry it seems that I´ve misunderstood your suggestion. I like your idea to test if there are still bounded interaction partners at the beads by a more stringent elution procedure like boiling in SDS.
Thank you so much guys!! Actually Murat, after eluting the complex with 100ul of this rehydration buffer, I'm running 10ul of this for western (as I have to use rest of the eluate for 2DGE). Probing with the antibody I use for IP, I can see band for input (raw sample used for IP) but not any band in IPed sample. Eve, I think I should go with your suggestion of boiling the bead and do western. Thank you.
Hi Kamalika,
Before you do 2D gel, make sure that your IP protocol work, by using 1D SDS PAGE and western blot as suggested above. Then you can move to 2D gel. After elution from the bead, you should first precipitate your eluate with aceton -20oC overnight before running IEF. By this way, you could eliminate some detergents/salt which are not appropriate for IEF. Good luck!
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