Isolation of PBCV virion proteins
Reagents
Liquid (88%) phenol, best quality by adding water to crystalline phenol
1% w/w Ficoll 400
0.5M DTT solution (77mg/ml)
10M LiCl
10% w/v SDS
1% Ponceau S
Acetone
Procedure
To 100ul of PBCV in a 0.5ml conical plastic centrifuge tube add 150ul of phenol. Add DTT (~10ul), 10%SDS (~20ul), 10M LiCl (~15ul) and Ponceau S (~10ul). Heat 5 min at 75C, mix thoroughly. Allow to cool, centrifuge 1 min (5K), draw off lower (clear) phase into a 3ml Pyrex tube. Add 1ml acetone and then 5ul of 1% Ficoll, mix, store 20 min at -20C, centrifuge 1min (6K). Discard supernatant, drain tube, wash ppt once with acetone (ppt sticks to tube) and dry. A hair drier speeds drying. Dissolve pellet in cracking buffer (~50u>l).
