We use wet blot transfer for our proteins the molecular weight of which is 18 -19 KDa. We use PVDF membrane for transfer and tris glycine SDS buffer contg methanol 20%. Our gel is 15% Stacking and 5% resolving gel 10 x 10.5 cm 1.5 mm thick.

The bands on the membrane are faint and no bands remain on the gel. We also get some additional high molecular weight bands on the gel which we suspect to be dimmer, but sometimes it is not visible clearly and sometimes we get good result. Is the transfer not efficient. We use alkaline phosphatase NBT and BCIP for detection.

Can anyone guide me on a good protocol for western blotting wet transfer method using NBT and BCIP. Also what condition shall I use for transfer since we do overnight transfers, can I can reduce the time

Also can I store by antibody used once and reuse it second time.

When you do the transfer you can try to change to Nitrocelulose membrane, some times it is better. Another posibility is to use a specific antibody instead of NBT and BCIP. The antibody way is much more sensitive. Are u using constant current for transfer? If yes how many mA ?
Write me more about the procedures and I will try to help you.

We use wet blot transfer for our proteins the molecular weight of which is 18 -19 KDa. We use PVDF membrane for transfer and tris glycine SDS buffer contg methanol 20%. Our gel is 15% Stacking and 5% resolving gel 10 x 10.5 cm 1.5 mm thick.
Run is 30 V, 160 mA, 30W, voltage constant, overnight run at 8 C
procedure followed after transfer
1. gel equilibrated transfer buffer 20 mins. PVDF membrane wet in methanol 1 min equilibrate in transfer buffer 10 min.
2 run overnight Bio-rad wet transfer
3.wash membrane with water
4. ponceau stg marking markers. destain.
5. blocking in 5%skim milk in PBS contg 0.02 %sodium azide11/2 hr
6 primary monoclonal Ab 2 hr
7. wash with PBS 3 x 10 mins then wash with Tris Nacl buffer 10 min
8. secondary Ab in tris NaCL buffer 1 hr
9. wash with Tris nacl buffer 4 x 10 mins
10. Development with Alkaline phosphatase contg NBT BCIP
The bands on the membrane are faint and no bands remain on the gel. Is the transfer not efficient. We use alkaline phosphatase NBT and BCIP for detection.

Can anyone guide me on a protocol for western blotting wet transfer method using NBT and BCIP. Also what condition shall I use for transfer since we do overnight transfers, can I can reduce the time

I would use a 30 min 100V (constant) for transfer. Then Rinse in TBS+tween 20 (0.05%) for a while. Block with 5% milk in TBS-T for 30 min and primary antibody over night at 4C on a rocker (slow speed). Wash 3x10 min in TBS-T and secondary antibody in 5% milk TBS-T for 1 hour at room temperature. Since your bands appear weak, maybe using an HRP conjugated secondary in combination with ECL. I use the one from Pierce and it works great. I wouldn't reuse the antibody, at least not until I got the method to work.

I would also try to use a much more sensetive WCL reagent. I.e. Pierce femto or Pico.
Both of them are very good to detec even a very faint signal.
Do a very quick exposoure.
Guy