Hi, all
We are acquiring a Western blot system soon and although I have always used the traditional wet transfer method, there are some recent equipment promising a much faster, less cumbersome and more effcient transfer (no liters of solutions and transfer in about 5 mins!!).
The disadvantages that I have found so far are:
1) limited to transfer of 11 kDa to 220 kDa - no problem for me
2) the need to use custom sandwiches - meaning we get tied to whatever they want to charge for them
Could someone share their experience with any of these systems? Are they as good as promised? Much more convenient than the wet transfer?
Thanks in advance!
We have the turbo blot system from Bio-Rad in our lab, It is much more convenient (time and effort wise) to use these transfer systems. A tank transfer usually takes anywhere from an hour to overnight at low V.
They do have pre-loaded protocols to use for 1 vs 2 gels in a transfer cassette etc. 99% of the time, they work fine. But with any generalized protocol , there are times where it doesn't for something very specific. For example. We had to troubleshoot the exact transfer conditions for some hard-to-transfer protein conjugates to detect them by WB. But you will face these problems with any equipment in specific cases.
Another possible con might be that you would have to use the custom made sandwich paraphernalia that comes with the specific company. We once switched to a cheaper/thicker blotting paper for the sandwich and the transfers didn't work as efficiently as with the company recommended blotting paper.
Hi, Archana! Thank you for your tips!
Yes, the problem with the Thermo seems to be that there is no alternative to their custom made sandwich.
Allow me a final question... On the Bio-Rad system would I be able to use any kind of membrane, buy the recommended filter paper, and reuse the rest of the components of the sandwich? (that would allow for some flexibility)
Thanks!
The membrane that we use is specifically PVDF and the three labs I've worked in so far all used PVDF, I think it wouldn't be advisable to use nitrocellulose, since the transfer buffer contains alcohol. We generally just buy the roll and cut it to size. Another thing to consider would be that the buffers also come prepackaged, although we've never seen any problems when we use homemade buffers.
Another thing that escaped my attention is that we've always used Bio-RAD tris glycine gradient gels. I'm not sure how the transfer would be affected if someone were to use homemade gels. Apart from the gel and the membrane, you only need the filter/blot paper to complete the sandwich. There's no need for the sponge that one has to use with tank transfer sandwich.So the only other consumable apart from the gel and the membrane is the filter/blot paper.
Thank you so much, Archana! That makes the whole thing much more clear to understand! Bio-Rad's system surely seems much more flexible than Thermo's.
We'll now consider if the difference in price is worth the convenience and time saved!
I have used Bio-Rad system with home made gels and PVDF membrane. It is very easy and works fine. We tried PVDF membrane from other companies, but it resulted in high background. We haven't tried to optimize the conditions for other PVDF membranes. So, we stick to the Bio-Rad kit with home made gels. I can recommend this system. Good luck!
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