PVDF vs. Nitrocellulose for Western blot - (Jan/31/2009 )

I'm quite familiar with using nitrocellulose for my western blotting and would frequently freeze the blots and reuse at a later date if necessary. I've switched to PVDF recently and have been having problems (hig background, inability to detect any bands). I suspect that some of my problems are due to freezing the membrane.

Does anyone have experience in this respect.

Also, I'd appreciate information on the differences between the two and advice on reducing background with the PVDF.

Mr Jefferson on Jan 31 2009, 02:07 PM said:

I would still go for PVDF.
Nitro is good when the bands that you get are strong. if they are faint you can hardly see them on Nitro.

when I develop the PVDF membrane with NBT+BCIP+AP Buffer, for the first 10-15 minutes I can only see the strong bands. but when time goes by I can see many faint bands in the background which still can be my result. this happened to me many times.

some people on this forum suggested activation of PVDF membrane by soaking it in pure Methanol for 1 minute prior to adding it to Towbin's buffer. it works fine for me now.

You really don't need to freeze blots to save them. In fact, I've never seen this done. Short-term (a week or two) you can keep the blots in TBST or PBST at 4 degree but be sure to change the buffer after one week. For longer periods of time you should dry your membranes (30 mins-1hr in 37degree incubator or overnight on your bench). I then seal my membranes in plastic and store them in my notebook with the films. This way I can always go back and rehydrate and reprobe.

You always need to hydrate PVDF with methanol and rinse with water (deionized) until it doesn't bead before transfer. Otherwise proteins don't stick quite as well. This might be part of your problem. What does the ponceau look like after transfer? Do you have good transfer?

For the difference between the two membranes, do a little internet searching of nitrocellulose versus PVDF. There is much literature and reasoning for each membrane and why they are good in certain instances. It would be best for you to do this reading on your own to learn about these two options.

Some antibodies show a preference to the membrane and will give higher background on one. I've never had issues not seeing bands but I have noticed PVDF requires more extensive washes than nitrocellulose to get rid of background. Also, some antibodies prefer TBST over PBST or visa versa. I am working with an antibody that is hideously dirty with TBST and gives perfect clean bands with PBST. It also seems to greatly prefer PVDF.

If background is still an issue you can do a second block between the primary and secondary antibody with the serum of the species that produced the secondary (usually goat). For difficult antibodies, I block in 5% goat serum before the secondary. This really helps bring down the background from the secondary antibody.

Mr Jefferson on Feb 1 2009, 09:07 AM said:

PVDF (especially sequencing grade PVDF, which is basically a double-thickness version) should give better results than nitrocellulose. It should bind more protein, as well as allow less blow-through. The only tip I've heard of is to prepare with MeOH, as others have said. If you're concerned about blow-through, reduce your current a bit.

I usually dry the blot by placing in between two sheets of grade 3 whatman paper. Once the blot dries, place the blot inbetween two fresh sheets of whatman grade 3 and place in 4 degrees C. I got good signals for about 1-2 months after that.

I think you could freeze them but make sure you dry the blot properly. Also make sure you wash teh blot in dH20 before drying. Maybe the prsence of water in the blot can somehow damage the protein or Abs while freezing and then thawing.


PVDF membranes are activated by methanol, so you can also try to rinse them in methanol after you take it out the freezer. Not sure if you should rinse off the methanol with dH20 after that but you can try both and see what happens

I have been using PVDF membranes for more than 3 years now, and its very fine. after I develop the membrane by ECL reagent or millipore regant and filming it, I store it at refregerator (for days) until I strip it for the next antibody. For blocking you can use either 3% skim milk for 1 hr at RT, or 10% skim milk for 10 minute at RT.

Mr Jefferson on Jan 31 2009, 09:07 PM said:

Ok, so it turns out my problem was the primary antibody. I had to go to a 1:10,000 dilution!

Thanks for the advice. I've swithed from freezing to just storing in TBST for a week and replenishing the solution every week if I need to keep them.

Just curious, what was ur starting dilution before?

Mr Jefferson on Mar 19 2009, 05:24 PM said:

Another difference between PVDF and Nitrocellulose is that PVDF is much more sensitive to SDS and may even loose of the already bound proteins if there is more SDS in the transfer buffer. Either do not use the SDS or keep it limited to less than 0.05%.

can't you avoid loosing protein from the PVDF by adding glutaraldehyde (0.05%) in your first wash after transfer?