This protocol can be used for samples for 2D or 1D IEF

Protocol for Urea Lysis
Urea lysis buffer
8-9M Urea, 5mM EDTA, 5mM EGTA, 1% DTT, 2-4% CHAPS
(Should be prepared freshly)
Washes
Cells should be wased 3 times with Ice cold PBS.
Last wash with 20mM Tris , 250mM Sucrose
Lysis
cells should be lasate with volume coresponding the diameter of the dish with the urea lysis buffer .
lyse at room temperature for 20 min (evry 5 min vortex quickly)
Spin down pelle. Transfer sup to clean tube.
save 10ul for Protein Assay
Samples could be freezed at 80 for later on running on 2D or IEF 1D.