I just switched from nitro cellulose to PVDF but I am facing some problems:

1- after 1 hour of transfer I can still see my pre-stained protein marker is on the gel. but when I use Nitrocellulose almost all the protein marker is transfered onto the membrane.

I know that I can use pounceau red to figure out if I've got any protein transfer at all or not, but unfrtunately I don't have it in the lab.

2- I usually set the power on 15V - 60mAmp. When I use nitrocellulose the current V and mAmp are usually lower than what I set. they usually stay on 3-4V and 60mAmp.....but now that I am using PVDF the voltage immediately goes to 15V.

why am I seeing less transfer while using PVDF?

have you activated your pvdf by soaking in absolute methanol immediately prior to use (without allowing it to dry)?

your pvdf may be acting as an insulator instead of allowing the current to flow if it is not properly wet.

Soak your PVDF in 100% methanol for 1 min on a shaker.

Rinse the PVDF in dH20 for 2 min on a shaker.

Place your PVDF and filter pads in transfer buffer while your gel runs (30-60 min).

NEVER LET YOUR MEMBRANE DRY OUT before you make your sandwich.

Transfer at 200 mA for 90 min.

Are you using 20% Methanol in your transfer buffer? If not, pump up the Methanol in your transfer buffer (provided your concerned with proteins under 100 kd).

Let your gel hand out in transfer buffer for 15-30 min on a shaker prior to transfer (provided your concerned with proteins under 100 kd).

Rinse out your tranfer tank after running the gel well to get rid of excess SDS.

Also check the Ph of your Transfer buffer, should be 8.3.

Thanks guys,

I didn't put it in methanol the first time, I think the problem was Methanol activation. in the next attempt I got a lot of protein transfer and sharp bands