Despite carefully assembling the sandwich structure (8% SDS-PAGE gel to nitrocellulose membrane) and using a Teflon roller to remove bubbles, I consistently observe minor bubbles after overnight wet transfer (25V, 300mA, 4°C). These artifacts compromise my results.
A proper setup would never introduce any bubbles during sandwich setup. Here is how I do it. I pour enough transfer buffer in a tray so that I can keep the whole cassette submerged within buffer while putting foam, paper, gel and PVDF membrane. While putting these one by one, I keep the cassette submerged within buffer by pressing with fingers. And most important, I soak and wet the foam and paper well by dipping into transfer buffer, press thoroughly few times to remove any bubbles stuck in the foam while dipped. Use roller to remove any bubbles get in between layers. if you keep the cassette beneath the surface of transfer buffer during setup, there should not be any bubble. Best of luck.
Hello, Chloe Sun.
The most likely cause is that bubbles were not completely removed from between the gel and membrane during the transfer process.
When performing a wet transfer, it is necessary to perform four bubble removal operations:
once after placing the filter paper, once after placing the gel, once after placing the membrane, and finally once after placing the top layer of filter paper. Make sure to squeeze the bubbles in one direction, not back and forth.
For gradient gels, bubbles should only be squeezed horizontally, not vertically. Also, make sure that the glass plates and spacers used to prepare the gel are thoroughly clean.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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