Phenol extraction of proteins
To 100 µl of PBCV - 1 in a 1.5 conical plastic centrifuge tube add 150 µl of phenol .
Add DTT ( ~ 10 µl ) , 10 % SDS ( ~ 20 µl ) , 10M LiCl ( ~ 15 µl ) and Ponceau S ( ~ 10 µl ) .
Heat 5 min at 75°C , mix thoroughly .
Allow to cool , centrifuge 1 min ( 5K ) .
Draw off lower ( clear ) phase into 1.5 conical plastic centrifuge tube .
Add 1 ml acetone and then 5 µl of 1 % Ficoll , mix .
Store 20 min at - 20°C .
Centrifuge 1 min ( 6K ) .
Discard supernatant , drain tube , wash ppt once with acetone ( ppt sticks to tube ) and dry .
Dissolve pellet in ~ 50 µl cracking buffer ( 1 % SDS ; 5 % glycerol ; 0.03125 M Tris - HCl , pH 6.8 ; bromophenol blue - trace ) .
