Problem with Western Blot Transfer Conditions?

[Renno]

Hello for everyone!
I'm new on this Forum.
I'm using PVDF membrane, and I'm doing the right treatment, whit methanol (30 seconds), water MilliQ (1 minute) and transfer buffer (10-15 minutes).
When I do the Ponceau S test after the transfer, the bands don't show up!!!
I'm think I'm having a problem whit the transfer!!
Here's my conditions to transfer the proteins:
- For 1 gel: 100V and 200 mA for 2 hours at cold room. But after 30 minutes, the V falling down to 40-50 V.
-For 2 gels: 140 V and 400 mA for 2 hours at cold room. After 30 minutes the V falling down to 65-70 V.

What is my problem? Is the V???
What do you suggest me? transfer overnight whit a low V (30V)?
Thanks for help me

[admin]

Hi Renno,
have you tried a fresh newly made transfer buffer? Seems that may be the issue or your transfer apparatus.

I would transfer overnight at 30V also, much better as you are transferring at high voltage but you mention it falls to 40-70V which is not enough after 30 minutes for a good transfer. Seems your buffer is having issues.

[Anastasia_S]

I transfer at 70V for 12 h. Now you made me think I'm overdoing it!

[Renno]

Hello again, and thanks for answer me.

Well, I chance some products of my transfer buffer, and the way that I make the "sandwich step", and now the thinks are ok again!!!
I think the problem was in the transfer step! Thank you Admin.

Admin, to transfer 30v overnight, what mA should I need to use?

Anastasia_S, my transfer stills 60 V (55-60v), 200 mA (for 1 gel) for 2 hours on ice, and I think it's enough time to transfer. I didn't have problem anymore.
Maybe for protein whit high molecular weight, you'll need more time. I can transfer in 2 hours, proteins with 70-72 kda for example.

See you soon!!!

Renno