The protein was transferred from the gel to the PVDF membrane and blocked with 1% BSA at 4 °C overnight.
Briefly, equal amounts of protein from each factor were separated on a non-reducing 12% SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% fat free milk in TBS/0.05% Tween 20 for 2 h.
Proteins were transferred to a PVDF membrane and blocked overnight in blocking buffer (Sigma-Aldrich).
The proteins were transferred to a nitrocellulose membrane and blocked for one hour in 5% non-fat dry milk.
Proteins were transferred to nitrocellulose membrane and blocked in 3% low fat milk in TBST for 1 hr.
Plasminogen overlay: 2-D gels were transferred to polyvinylidene difluoride (PVDF) membrane and blocked in PBS, 5% blotto.
Samples were loaded into a 7% polyacrylamide gel, transferred onto PVDF membrane and blocked for 1.5 hours in 5% dry milk∶TBS-Tween solution.
Proteins were then transferred onto nitrocellulose membrane and blocked with 5% non-fat milk in PBST (1× PBS and 0.1% Tween-20) for 1 h at room temperature.
The samples were run via SDS-PAGE on a blank gel, transferred to a nitrocellulose membrane, and blocked with antibodies against Hsp90α.
Proteins from lysed cells (15 µg) were separated by 10% SDS-PAGE, electrotransferred to nitrocellulose membrane and blocked with 4% skimmed milk in PBS.
Equal amounts of protein were separated on a 4 12% Bis-Tris gel (Invitrogen), transferred to a nitrocellulose membrane and blocked in TBS containing 5% skim milk and 0.1% Tween 20.
