To conduct my western blot, I used a PVDF membrane which I activated by wetting the membrane in methanol for 1 minutes ( change from opaque to translucent), placing the membrane in Milli Q water and soaking for 2 minutes, then placing in transfer buffer (without methanol) to equilibrate for 5 minutes at least and I quickly placed the membrane to assemble the western blot to prevent the membrane from getting dry.
(Note: the recipe for my transfer buffer is 3.03 g Tris and 14.4 g of glycine which I then added dH20 to make up 1L)
As you can see in my photos, after transferring the proteins overnight (around 16 hours), I found that the protein ladders and samples transferred onto the filter paper instead of the PVDF membrane. Even more strangely, when I dyed the PVDF membrane with ponceau staining, I was able to visualize a clear outline of the shape of the gel.
I was wondering if anyone had an idea of what could have possibly went wrong?
Thank you!
Hello..
This discussion would be helpful to you.
If you want to use PVDF membrane you have to use "METHANOL" in 1X transfer buffer, Check your transfer buffer recipe. More than that if you want to do Overnight transfer reduce the voltage to 40.
https://www.researchgate.net/post/I_want_to_run_overnight_western_blot_electrotransfer_what_voltage_should_I_use_The_protein_is_p-ATM2
I agree to dave, I think the transfer voltage was possibly to high for a 16h transfer.
Possibly the membrane is not suited for your western blot (pore size etc.).
Moreover, I would propose to revise your protocol, especially regarding solutions.
A potential source for optimal working protocols is https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=15994
Hello Andreas Eisenreich and Dave Unis and Ramesh Mariappan . I run my overnight transfer at 30V. I will try lowering the voltage. Thank you!
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