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文献和实验9M Urea,2.5mM EDTA,2.5mM EGTA,1% DTE,4% CHAPS make 10ml and aliquot 10x1ml,freeze at -70℃ Lysate preparation wash the cells 2x with PBS wash the cells 1x with 10mM Tris,250mM Sucrose lyse the cells with 100 – 350μl of urea lysis buffer
ULTIMATE FREEZE-THAW LYSIS FOR MAMMALIAN CELLS
tube.3.Pellet cells at top speed in microfuge (45s in cold-room).4.Resuspend cell pellet in FT LYSIS Buffer (25-40 μl per confluent 6 cm dish,50-80 μl per confluent 10 cm dish).Resuspend by pipeting up and down.5.Drop tube in liquid nitrogen to freeze
Preparation of Bacterial Proteins for Analysis by 2D-PAGE
to the bacteria under study the following buffers are used. 2D Lysis Buffer 0.01M Tris-HCl, pH7.4 1mM EDTA 8M Urea 0.05M DTT 10% (v/v) glycerol 5%(v/v) NP40 6% (w/v) ampholytes The buffer is made up using high purity MilliQ
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