Preparation Of Whole Lysate From Brain Tissue

Preparation of whole Lysate From Brain Tissue (For Western Blotting)

Lysis Buffer:

Final Concentration Stock Solution Prepare 200ml Prepare 50ml (PH=7.4)

50mM Tris-HCl 250mM Tris(PH=7.5) 40ml 10ml 150mM NaCl 5M NaCl 6ml 1.5ml 0.25% SDS 10% SDS 5ml 1.25ml 0.25% Sodium 10% Sodium Deoxycholate Deoxycholate 5ml 1.25ml 1mM EDTA 500mM EDTA 0.4ml 0.1ml

Add 1-10% Proteinase inhibitor Cocktail (Sigma P8340) Before use

Protocol:

1 ml Lysis Buffer + Tissue  Passing through 18G needle( 10 times) + polytron 10 times(50% setting)  Vortex 5 times during 30 min on ice  10,000rpm ( 10,400 g)x 10 min on 4C  Keep Supernatant (Whole Lysate)

* Cell Pellet: --- aspirate medium from cell plate --- add 2ml ice cold PBS, then aspirate it --- add another 2ml PBS, scraple cells off the plate, transfer cells to 10 ml tube, keep on ice --- 1000xg centrifuge for 5 min --- discard supernatant, keep cell pellet at -80C