Lysates were boiled for 3 min, resolved by 10%% SDS/PAGE using Tris-glycine pH 8.3 (25 mM Tris, 192 mM glycine, 0.1 % SDS) as running buffer, and then electroblotted onto a Hybond nitrocellulose membrane (transfer buffer: 25 mM Tris, 195 mM glycine, 0.05 % SDS, pH 8.3, and 20%% v/v methanol).
Approximately 40 μg of protein extract was separated by 8% SDS‐PAGE and then transferred to nitrocellulose membranes (transfer buffer: 25 m m Tris, 190 m m glycine, 20% methanol, 0.5% SDS).
Proteins were electro-transferred to NC membrane in transfer buffer at 100 V, 4 °C for 1 h.
Immobilon PVDF membranes (Millipore) were prepared by wetting the membrane for 15 seconds in methanol, equilibrating the membrane in distilled water for 2 minutes, and then equilibrating the membrane in transfer buffer (25 mM Tris base, 10% methanol (v/v), 192 mM glycine) for 5 minutes.
The samples were separated in a 7.5% gel and then transferred to an Immobilon-P PVDF membrane in transfer buffer.
Equal loads of proteins (20 µg) were subjected to electrophoresis in 9% polyacrylamide gels in the presence of SDS and the resulting bands were blotted onto a nitrocellulose membrane in transfer buffer at 50 mA overnight.
The gel was transferred to a polyvinylidene diflouride membrane in transfer buffer (25 mM Tris base; 190 mM glycine; 20% [vol/vol] methanol; pH adjusted to 8.0), fixed in methanol, air dried, and blocked with TBS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl) containing 0.5% Tween-20 and 5% (wt/vol) nonfat dry milk (NFDM) for 1 2 h at RT.
Proteins were transferred to a PVDF membrane in transfer buffer (Invitrogen) at 25 V for 2 hours.
Protein was then transferred to a PVDF membrane in transfer buffer (3.7% SDS, 20% methanol, 48 mM Tris base, 39 mM Glycine) at 4°C.
Aliquots of the treated lysates containing protein 50 μg/ μL were electrotransferred onto the PVDF membrane in transfer buffer for 1 hr.
Samples containing 20 40 μg protein were loaded on a 10 12% SDS PAGE gel and then electrophoretically transferred to a polyvinylidene difluoride membrane in transfer buffer (25 m M Tris, 190 m M glycine, 20% methanol).
