PVDF membranes are great for Western blots. They bind protein readily, can take a beating from a strong-handed researcher and are ideal for fluorescent Western blots.
But they do have an annoying quality that adds an extra step to your protocol: they have to be activated prior to use.
Activation is important
If you use a PVDF membrane for your blot, then you have to activate the membrane by soaking it prior to use. Most protocols recommend wetting the membrane in 100% methanol for a few seconds and then equilibrating the membrane in transfer buffer for a few minutes (until it sinks). Some protocols also say to equilibrate the membrane in water before transfer buffer.
Don’t skip the activation step. It is important.
Unlike hydrophilic nitrocellulose membranes, PVDF is extremely hydrophobic. So hydrophobic in fact, that aqueous buffers, such as transfer buffers, will not be able to penetrate the membrane. Without activation, proteins will not transfer and bind to the membrane easily. Methanol hydrates the membrane increasing the protein-binding capacity.
Do you have to use methanol?
Being the superstitious scientist that I am (why fix something that ain’t broke?) I’ve always used methanol for activation And I would bet that most researchers stick with methanol. But if you look closer at protocols, you will see that ethanol and isopropanol can both be used to activate the membrane.
So why all the fussiness? It seems to come down to purity and time.
Purity
You always want to use high quality alcohol for membrane activation. Impurities and solvents in alcohol can mess up your transfer. Buying pure ethanol can be tricky. Even reagent grade ethanol can have up to 10% of solvents in it! If you are going to use ethanol, you need to buy 200-proof or molecular biology (non-denatured) grade.
Time
If you are going to use an alcohol other than methanol to activate the membrane, then add a few extra minutes into your protocol. If you use the water step, you will need more time to equilibrate the membrane with water. Ethanol/water mixtures are more viscous than methanol and it will take longer for the ethanol to diffuse out of the membrane. In addition, both ethanol and isopropanol are more hydrophobic than methanol, so you will also need to equilibrate with the transfer buffer for a longer time.
Some tips for activation
Whichever alcohol you choose to use, here are few tips to make your transfer more successful:
- Do not allow the membrane to dry out after wetting – a dry membrane will not bind protein
- If the membrane does dry out, you can rewet it by following the activation steps again
- Don’t try to activate a nitrocellulose membrane in alcohol – you won’t like the result
- You can use 50% methanol for activation, but increase the activation time
- Always use gloved hands when working with the membrane
Photo courtesy of Vicente Villamón
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