Protocols:

 

Cell Lysis & Extraction

 


Lysis Buffer:
20 mM Imidazole-HCl, pH 6.8
100 mM KCl
1 mM MgCl2
10 mM EGTA
0.2% (v/v) Triton X-100
10 mM NaF
1 mM Sodium Vanadate
1 mM Sodium Molybdate
10 mM Sodium Pyrophosphate
25 mM /td> b-Glycerophosphate
Aliquote and store frozen!

Use 0,75 ml Lysis Buffer per petri dish of 100 mm diameter. Perform cell lysis at room temperature under optical control until complete.
Clear spin cell extract to remove nuclei (5 min, 12000 g, 4C) and recover supernatant.
Immediately add 0,25 ml 4x SDS-PA-GE sample buffer to 0,75 ml supernatant, heat to 90C for 5 min. Aliquote extracts and store frozen if not used immediately. Avoid repeated freeze/thaw cycles.
Apply about 20 l per lane on a Biorad Minigel (amount should correspond to 1-2 x105 cells per lane).
It is important to load lysates of equal numbers of cells on each lane. Due to the high protein content of the sera, a protein determination is not useful. Individual samples can be calibrated by immunoblot using another antibody. We strongly recommend to use the high affinity monoclonal antibody MAPK2-6G11 directed against the C-terminus of MAPK2 for calibration of Blots.

Mouse