preferably in transfer buffer. I don't soak NC membrane for long. As long as the membrane is fully wet before putting it in the transfer sandwich, you'll be fine.

preferably in transfer buffer. I don't soak NC membrane for long. As long as the membrane is fully wet before putting it in the transfer sandwich, you'll be fine.

I soak in cold transfer buffer, generally 5 minutes is fine. I usually start soaking the blots while I prep the gels. It is also helpful to soak your filter paper and sponges. 

That should be plenty of time. Nitrocellulose wets very quickly. With PVDF, you have to pre-wet with methanol - so you need more incubation time in transfer buffer to equilibrate the buffer systems.

I agree with Bing that it's best to wet the membrane in transfer buffer so that everything in your transfer stack is equilibrated with the same buffer system.  

Many transfer buffers contain SDS, and this can cause trouble with bubbles when setting up your transfer stack. You can soak the membrane in transfer buffer without SDS added, to eliminate this problem.

Transfer Buffer with MeOH

in my opinion, you should soak your nitrocellulose in your transfer buffer.  I soak mine for 10 minutes as well.  I also soak the blotting paper in transfer buffer for 10 minutes.

I also agree with Bing Yu. I just put the dry membrane on the gel, make sure there are no bubbles and it stick well, next fill the dish with buffer and add well soaked filter paper and a scouring pad. it always works

transfer buffer is usually SDS free

I also soak the membrane first in methanol till it becomes wet (mostly for pvdf membranes), then prepare the cassette fully immersed in Transfer buffer which also contains methanol. For nitrocellulose membranes, you can directly put in transfer buffer for few minutes before preparing the cassette. Also the sponges need to be wet in transfer buffer. After your cassette is complete, put the roller on the top gently to avoid the bubbles. And yes, the transfer buffer that we use contains TRIS-Glycine, SDS and methanol. For blocking and washing the membrane after Western blotting, we use TBST or PBST. Both works fine.  

I agree with the other suggestion. I soaked the nitrocellulose, sponge and 3MM in cold transfer buffer (with MetOH) then I unplugged the gel from the glasses. For this purpose I put the soaked 3MM on the gel to separate it from the glass, without consequences, and I soaked, for few minutes, the gel in the cold thansfer to eliminate the wrinkles at the bottom of the gel.

Hi bonnie

generally speaking you don't need any incubation before hand if you are using nitrocellulose membrane. Nitrocellulose is less hydrophobic then PVDF. So even a minute in TRANSFER BUFFER ( MeOH) will do. Now transfer buffer don't have SDS or any other detergent, it is good if you avoid putting any detergent ( TWEEN in TBST) with soaking your membrane in TBST. However some people use trace amount of SDS in transfer buffer especially when their target is High molecular weight.  

All of these are very good answers.  Since nitrocellulose membrane does not have to be activated like PVDF it only needs to be wetted using cold transfer buffer.  I have noticed that the wetting process does make a difference on the western blot results (i.e., very little background). 

You will notice from the image below that the wetting process in from left to right or right to left (doesn't matte).  The point is make sure that it is wet evenly and you do not handle the membrane excessively.

Ahmed..you do not need to soak the nitrocellulose membrane in pure methanol...the nitro cellulose will  equilibrate in the standard transfer 20% MeOH, Tris-Glycine buffer.

Was there a compelling reason to soak the membrane in the Methanol other than to wet it before placing it next to the gel?

With PVDF, after 30 sec to 1 min wetting in methanol..we need atleast 20 min in transfer buffer ...otherwise the transfer is not efficient. I did one with 15 min, it was very poor. Nitrose clellulose wets quickly.. It is recommended to soak the gel after run in TBS, didnt try though without soaking.

Unfortunately i didnt wash my membrane after 5 min soaking with methanol.I didnt get any band after transferring including ladder.Is there any methanol effects for transferring the protein?

Transfer buffer does not have any SDS but it contains Methanol. Good practice is to soak the membrane around 5 min before making the sandwich.

If you are using PVDF membrane, then you soak the membrane in 100% methanol for 15 minutes and one more think, don't add SDS in TB if your target is low molecular weight protein, otherwise you can add.