I am trying to run 2D gel. I am extracting protein from mammalian cell by using RIPA lysis buffer and then precipitate the protein with acetonitrile, then re suspend the protein in 250μl of re-hydration buffer sonicate the sample for 10mins and then start IEF focusing and so on by using invitrogen protocol and this is what I get every time. Can someone suggest me any solution on how to improve the gel or the whole procedure?
Hi Faruk,
I'm not sure what the 'Invitrogen protocol' that you're referring to entails, but you could try to reduce the horizontal smearing that you observe by running your first dimension (iso-electric focusing) longer/differently.
Generally, that run would consist of starting at a low voltage for a relatively short period of time to get rid of residual salts present in the sample. Then the voltage is ramped up to a relatively high voltage to enable protein focussing. Once the current you observe over your gel becomes constant, an equilibrium should've been reached, which means that your focusing is complete.
Hope this helps!
Thank you for your suggestion Dirksen but I have already tried that, the image I shared was after applying that suggestion. Thank you again.
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