PVDf membrane activation....

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PVDf membrane activation....

Postby Jack Hilton » Aug 04 2007 6:19 am

Hi everybody,
i routinely use PVDF membrane to transfer proteins in western blot. To transfer the proteins, first i have to activate this PVDf membrane with methanol, logic behind this step i didn't understand. Can any tell me why this step is necessory and what is the exact role of mehtanol in membrane activation ?
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Postby relaxin » Aug 04 2007 11:00 am

I think it is to wet the membrane, otherwise the membrane is too hydropbobic to interact with the aqueous solutions.
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Postby melwisc » Aug 05 2007 9:25 am

Relaxin is correct.
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Postby witch_doctor » Aug 07 2007 1:04 am

Exactly so.
Methanol makes the pores on the membrane 'open' so as to facilitate protein binding. When using PVDF, you can decrease methanol in your transfer buffer, or omit it altogether, provided of course you have activated your membrane first. This allows greater binding efficiency than nitrocellulose.
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Thanks

Postby Jack Hilton » Aug 10 2007 8:29 am

Thanks !

witch_doctor wrote:Exactly so.
Methanol makes the pores on the membrane 'open' so as to facilitate protein binding. When using PVDF, you can decrease methanol in your transfer buffer, or omit it altogether, provided of course you have activated your membrane first. This allows greater binding efficiency than nitrocellulose.
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Postby r.rosati » Aug 10 2007 8:51 am

I use the same trick when I accidentally overdry DNA. Just add some more 70% ethanol to the overdried pellet (or 100% ethanol followed by water if the pellet is big), spin at max speed for 1 minute (to remove any air bubbles in the pellet), and air-dry again.
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Postby RRam » Aug 12 2007 1:37 am

To add in more, you can store the membrane after protein transfer simply by drying and covering with the filter paper. But before proceeding to further step like blocking, you should wet the membrane with methanol, followed by the water/buffer (to replace the methanol) which you would for the sucessive step.
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Postby witch_doctor » Aug 12 2007 3:20 am

RRam wrote:To add in more, you can store the membrane after protein transfer simply by drying and covering with the filter paper. But before proceeding to further step like blocking, you should wet the membrane with methanol, followed by the water/buffer (to replace the methanol) which you would for the sucessive step.


A tested procedure to store PVDF membranes:

1: Strip the membrane of any antibodies used so far, otherwise the antibody may become fixed when you dry the membrane
2: Dry it, place it between 2 whattmann papers, and cover with plastic wrap (the stuff used to cover food).
3: Store at 4 for a few months or at -80 indefinetly.

When you want to use it, dip it in methanol, then block it for 30-60 min, and you're ready.
I've tested membrane stored for 8 months, and the signal was perfect
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charge activation of PVDF

Postby Jack Hilton » Aug 18 2007 9:03 am

Thanks for this information.
But i have heard that methanol treatment makes the PVDF membrane positively charged , this allows negatively charged proteins to bind to the membrane.
Any comments about this.
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Postby RRam » Aug 18 2007 10:02 am

I dont think that methanol can make the PVDF positive charge as we are removing the methanol totally from the membrane before protein transfer. Perhaps my thinking here there is no reaction between membrane and methanol.
The Charge difference is mainly due to the current where you are keeping the membrane near to anode and the gel near to cathode.
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Postby mdfenko » Aug 19 2007 11:33 am

the methanol "wets" the pvdf membrane which allows the blotting solutions to interact with the membrane. binding is through hydrophobic interactions.
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Exposure time

Postby Jack Hilton » Aug 29 2007 12:49 am

Thanks

Genrally some people expose PVDF membrane to methanol for 30 - 60 sec and some people suggested 15 - 30 minutes exposure to membrane to activate it.

Why there is such a huge difference between exposure time ?

which exposure time i should follow ?
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Postby relaxin » Aug 29 2007 9:25 am

60 seconds should be long enough. But, some people may want an excuse to drink a cup of coffee or chat with the cute girl in the lab next door, they just soak it in methanol for 30 min. :D
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Postby RRam » Aug 29 2007 9:30 am

Jack,
Previously I have also confused with the timings like you. To my work I am currently using one few seconds of exposure and previously I have wet the membrane for few minutes say around 4 minutes. Both gave me the same result. From my work, here there is no time dependent factor. The main principle is to wet the pores as the membranes are hydrophobic nature. So you no need to worry about the exact timings.
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Re: PVDf membrane activation....

Postby peejay » Jun 20 2013 3:54 am

these are all *** answers. how can the PVDF membrane have "PORES" ??????? o_O
Atleast give some good reference along with all the supposed answers that pop in your mind.
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