I am looking for optimised protocol for western blot of 2D gels! any one routinely doing please help me!! I am missing minute details like voltage , buffer etc. Working wirth semidry GE healthcare transfer unit.
since the second dimension is basicaly classical SDS-PAGE I'd say you can use your standard protocol. But i have never done it (in fact I've ever heard before someone would do blot from 2D elfo).
Good luck!
The most complexe is to do 2D gel. After, the 2d dimension, do the western blot as a classical WB. You will have no problem to see your protein of interest.
In addition, you could use a control protein as a reference for pI and find the the pI of your spots. Becareful, GAPDH is a basic protein and often can not be seen in 2D western blot (due to the pHi).
2D Western blot works well !!!
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