7 replies to this topic

[almost a doctor]

Hello, I'm currently setting western blot in my lab (from having nothing), and I was wondering what are people's preferences on membranes and why.

Nitrocellulose or PVDF?  

all your thoughts will be welcomed    

[Roo]

I prefer PVDF.  Many years ago my lab was having trouble with our nitrocellulose so we switched to some PVDF we had stashed in a drawer.  The Westerns started working again so I have never switched back.  I don't know if the nitrocellulose had gone bad or if it just wasn't the best choice for our proteins at that time.  

We sometimes save our blots for stripping/reprobing at a later date.  We found that the nitrocellulose is more brittle so it falls apart during the stripping after being stored for too long while the PVDF doesn't have this problem.  

Some of the manufacturer's of these membranes have good product literature/comparisons on the different types of membranes.   I have used GE Healthcare/Amersham, Millipore, and others.  Below is a link to literature by Millipore.  Scroll down to the table that compares the membranes.  Maybe it will help you decide what is best for your applications.

Comparison of PVDF and Nitrocellulose Membrane Attributes and Applications
Millipore

[mdfenko]

we used small pore (0.2um) supported nitrocellulose for routine westerns and glycoprotein stains (chromogenic), pvdf for sequencing (edman degradation).

the support made the nitrocellulose strong enough and it was cheaper than pvdf (especially when buying by the roll).

Edited by mdfenko, 04 August 2009 - 12:29 PM.

[almost a doctor]

Thanks for your comments. I had heard before too that PVDF is more resilient and hence easier to handle, that supported nitrocellulose might be worth a try.

What about binding properties?  I'm intending to transfer and probe really small amounts of protein, so I guess my choice is going to be determined by that too... should I just try both?

[little mouse]

Then I would go for PVDF. But you might have more background with PVDF than with nitrocellulose (depending on the antibody you use, with good antibodies I don't have background with PVDF)

[mdfenko]

both have sufficient binding capacity for small amounts of protein. pvdf has more capacity than nc so would be preferred for larger amounts of protein.

[Mats_Nilsson]

mdfenko, on Aug 6 2009, 11:16 AM, said:

both have sufficient binding capacity for small amounts of protein. pvdf has more capacity than nc so would be preferred for larger amounts of protein.

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 idividual 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 - 10:47 AM.

[mdfenko]

smack...

see my reply to your other post...

Edited by mdfenko, 12 February 2010 - 11:32 AM.