We have been using XCell SureLock™ Mini-Cell Electrophoresis System with pre-casted for quite some time without any major setbacks.
Recently, we have been trying to perform electrotransfer of home-made polyacrylamide gels with the following specifications: Tris Glycine SDS PAGE gels 10% resolving gel, 1mm. The PVDF membrane is Millipore and the transfer buffer that we have been using is classic Towbin buffer Tris 25mM, 190 mM glycine and 20% methanol (sometimes with 0,1% SDS). The electrotransfer conditions are 35 Volts for 2,5 hrs.
We are not satisfied with the result of the electrotransfer process (judging from the Ponceau staining) and we are having problems with certain antibodies that used to work well.
I was wondering whether the transfer buffer needs to be adjusted or some other parameter needs to be changed. Personally i don't have much experience with semi-dry systems and I couldn't find anything pertinent over the Internet regarding this particular model and home-made gels.
Just use the transferbuffer from Thermo that is meant to be used with the Xcell SureLock in combination with your own self-cast gels. I do this as well and it works like a charm for me.
Transfer the proteins at 30v for 1.5hrs. with cold water in the outer chamber. I think 2.5hrs especially with a 1.0mm is way too long. Try 1-1.5hrs with your current protocol.
Assuming that you are using Xcell II Blot Module, I agree with Tim Schelfhorst.
I used to have lots of troubles with the blotting module when the transfer is performed in Towbin buffer. However, the blotter seems to work well with the proprietary NuPage Transfer buffer.
Thank you very much for your very crucial information. Just to be clear when you say transfer buffer from Thermo, are you reffering to the following product which is for pre-casted Novex polyacrylamide/Tris-Glycine gels LC3675 Novex® Tris-Glycine Transfer Buffer (25X)?
I was reffering to the NuPAGE transfer Buffer (NP00061) that I always use according to their protocol (50ml methanol for 1 gel, 100ml methanol for 2). It takes roughly 12,5ml of the 20x solution per transfer (1 or 2 blots).
Thank you very much for the clarification. To be honest I wouldnt have thought about it (because of the different composition of Tris-glycine gels compared to NuPAGE Bis Tris gels). We have this kind of buffer for our pre-cast bis-tris gels so I will use it with the home-made gels too.
I am glad to report that I followed your advice to the letter and I have managed to perform a good WB (major improvement compared to my previous efforts with this particular device). I used ~25 ug of protein lysates. I performed an electrotranfer for 1,5 hr at 30 Volt (NuPAGE transfer buffer, 160-138 mA, on ice with pre-cooled water in the outer chamber of the apparatus). Ponceau staining and WB development were spot on. When I stained the agarose gel with Coomasie some bands appeared but overall I think I have made considerable progress. Thanks a lot once more!
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