I am having consistent trouble getting my western blots to look good. Upon imaging the probed blot(s), I can clearly see bubble-like spots on the western blot. However, I roll the blot very well before transfering, and think the issue is not simply bubbles between the membrane/gel. (see picture)
I transfer at 100V for one hour (.3A) using the bio-rad mini trans-blot cell using house-made transfer buffer. For transfer, we use a bio-rad 4-20% gel as well as 0.45um PVDF-FL membranes.
My current thinking is that perhaps the power is too high, and the gel begins to melt mid-transfer, even though I pack ice around the transfer box. I haven't seen any visual change in the gel, but figure changes in the gel might stop efficient transfer. Any other ideas? Thanks for the help.
If you suspect power dissipation being too high (the poly acrylamide gel won't melt like agarose does but it will swell when it gets too hot), then simply half the voltage (or current), just keep U* I * time = const.
Did you check with Ponceau, to show it is actually a transfer problem?
If you are not doing this already, it helps to equilibrate the gel in the transfer buffer for a few minutes.
Or it could be poor activation of the membrane. Lower the membrane into the methanol slowly to displace air bubbles: drop one side in first then let methanol slowly rise along the membrane to the other edge.
Don't press the gel too much when rolling out bubbles or it may stretch and then shrink during transfer.
Also make fresh buffers and check the equipment is very clean, especially the cellulose pads (perhaps its time to buy new pads?) I've seen patterns from old pads appear on membranes.
I run my transfer in the cold room and with ice blocks.
This indeed looks like a transfer issue. And you can see where the bands have deformed around the bubbles, implying that there are in fact bubbles despite how well you may be rolling them out. I assume you are rolling gently and not vigorously rolling back and forth. How snug is your sandwich? Is the cassette firmly holding everything in place? Secondly, based on your description, it sounds like you are using constant voltage. How high is your current at the end of the run? Here, I am trying to rule out issues with the transfer buffer. How do you mix the transfer buffer? Are you introducing a lot of air using inversion or do you mix using a stir bar? My thinking is if you are sure you are rolling the bubbles out, then bubbles are forming during the transfer.
I do not think it is a problem of the Licor itself, but as you said, to your transfer.
As the bottom and the top of the membrane have a different size I suggest a swelling, but I do not believe that is the origin of your bubbles pattern on the membrane.
Like Brian I suggest formation of foam during the transfer because of to high temperature / presence of detergent.
I keep my transfer buffer at 4 degrees. I then put my pre-cooled transfer box into ice as you mentioned. I also do a quick wash after transfering (before blocking) because I worry about solubilizing protein off of the membrane. Speaking of membranes, are you using PVDF or nitrocellulose? If your protein is moving after transfer, you might want to try a different membrane type.
I agree with Sudhadeep if background is an issue. I've produced great looking blots with nitrocellulose. I've also used PVDF-FL which is also formulated to have low background on licor.
We use exactly the same set-up, Biorad gels etc. We experienced the same problems easily solved, go to your local Tescos buy some scotch pads cut them to size and use them to transfer your blots. Oh also make sure you have fresh transfer or transfer that has been kept at 4oC, do not use no more then 3X.
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