AG Daniel:Protocols

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AG Daniel

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Forschungshaus Charité
Lindenberger Weg 80
13125 Berlin

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protein analysis

Purification:

protocol recipe
x harvest cells
x wash with PBS
x lyse or freeze at -80°C 50mM Tris (pH 7,5) 0,33g
270mM Saccharose 4,62g
1% Triton X100 0,5 ml
ad dd H2O 50 ml
1 pill Complete Protease Inhibitor
aliquot and store at -20°C
x incubate on ice for 30min
x spin at max speed for 15min at 4°C
x transfer supernatant (=protein) in new tube, store at -20°C
x determine protein amount with BCA method
x prepare protein for loading (with 5x loading buffer) 300mM Tris (pH 6,8) 0,47g
50% Glycerol 5ml
2% SDS 0,2g
3% β-MercaptoEtOH 0,3ml
1 Sp Bromphenolblue
ad dd H2O 10ml



SDS-PAGE:
Western Blot:
Detection:

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