20 replies to this topic

[Mr Jefferson]

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.

[Curtis]

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

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.

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.

[rkay447]

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.

[swanny]

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

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.

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.

[MaggieRoara]

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

[yobou]

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]

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

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.

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.

[medchemgirl]

Just curious, what was ur starting dilution before?

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

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

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.

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.

[repeatcell]

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%.

[Tfal]

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

[proteo]

rkay447, on Feb 1 2009, 07:53 AM, said:

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.

Do you block after the wash steps right before you add the secondary? How long do you block for in the goat serum?

[fishdoc]

I've got a question about PVDF membranes. I've read a couple forums on the internet, but haven't found this in any descriptions or protocols for PVDF membranes. Following transfer, can you allow the membrane to dry, and then do the probing with your antibodies? I've read that you don't have to block if the membrane is dry. The reasoning is that once the membrane is dry, proteins will not bind to it (therefore, no antibody background) and the antibodies will only stick where the target antigen is on the membrane.

Has anyone done this or have any experience to say whether or not this would work?

[Feelcontraire]

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

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.

I think that the pros of PVDF is retention of small proteins and handling.
Nitrocellulose has the advange of no need for activation.

I usually store my membranes at RT in plastic bags.

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

Some ECL formulations don't like tween, so before adding the ECL do a wash on distiled water or PBS/TBS.
Never allow the membrane to dry during primary or secondary antibody incubations, the proteins would bind to the PVDF.
As you already did, reduce antibodies concentration.
You can try blocking with PVA.
Increasing blocking or tween reduces background, but may remove your protein from the membrane.

To improve detection limits don't use any protein blocking and reduce tween20 to 0,01% just block with PVA (130-180k) 1-100parts per million 1minute prior to primary antibody incubation. You can also microwave the membrane slightly in stripping solution to improve detection (before blocking).

I suggest activating in ethanol instead of methanol, it works equal or better and is enviromentaly friendly.
After this activation remove the ethanol by briefly shaking in water-buffer.

Letting the membrane dry after transfer may help to retain the proteins on te PVDF.
To reduce background perform short film exposure, to detect " the undetected" expose longer 2h-overnight.

Edited by Feelcontraire, 18 November 2009 - 08:42 AM.

[Mats_Nilsson]

Smack my face if I am wrong here: This is all ANECDOTAL EVIDENCE! Why is research littered with non-fact based opinions? There have been studies done by "electrophoresis/transfer studs" elucidating many of these matters (transfer buffer, wet vs. semidry, nitro vs. PVDF etc). Charged nylon appears to be great depending on the application. 0.2 um pore-size nitro has a higher binding efficiency than PVDF (presumably 0.45 um). Of course binding is dependent upon the individual protein itself, but in general there are some clear-cut info out there.

This is just one example and perhaps not the best (perform the PubMed search for other articles)

Electrophoresis. 1990 Jan;11(1):46-52.

Important parameters in semi-dry electrophoretic transfer.
Jacobson G, Kårsnäs P.

HERE IS SOME CONTRADICTING EVIDENCE (Remember that Pluskal has a patent on Immobilon + he washes the membranes after transfer 15 min at pH 10);

J Acquir Immune Defic Syndr. 1988;1(4):333-9.

Improved HIV antiglycoprotein antibody detection by immunoblotting on a hydrophobic membrane.
Lauritzen E, Pluskal M.



As a rule I would suggest a two-step wet transfer with 0.2 um nitro membranes as described by (the question is do you have the patience?):

Anal Biochem. 1987 May 1;162(2):370-7.

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins.
Otter T, King SM, Witman GB.

Everybody: Have a nice day and quit the anecdotal evidence thing. You are confusing new and upcoming students/researchers including myself.

Edited by Mats_Nilsson, 12 February 2010 - 10:49 AM.

[mdfenko]

smack...

what you are calling anecdotal we are calling experience. don't let the "studs" fool you, as you pointed out, some of them have vested interests in the results they report.

you can read all the papers you want (and we have) but nothing beats experience.

and, by the way, as you continue your career in science, you will find that many authors omit parts of procedures that may be important (even critical), intentionally or not.

so, when someone gives you the benefit of their experience it may be wise to, at least, take it into consideration rather than just write it off as "anecdotal".

Edited by mdfenko, 12 February 2010 - 11:34 AM.