Hello everyone,
I'm constantly struggling with one issue. I have to detect dystrophin (427 kDa) on western blots.
I use 4-15% TGX-PAAG and have tried dozens of different transfer protocols. Sometimes there is a band and sometimes there isn't, even if conditions seem to be unchanged.
My samples are usually mice-derived muscles (heart, triceps and so on).
Could you share your experience in this area? Have anyone determined a robust method for the detection of proteins >400 kDa?
Thank you in advance!
Hi Yulia,
I think you can give a try with SDS-agarose gel instead of SDS-PAGE.
Read these articles, you will get an idea about how to cast the gel and transfer.
Article Protein Electrophoresis in Agarose Gels for Separating High ...
If you can not access to previous article , read this one
Article Detection and quantification of the giant protein titin by S...
Read this too Article SDS Agarose Gels for Analysis of Proteins
You can add 0.5% of agarose to your polyacrylamide gel. The agarose has little effect on separation because its very large pore size, it only stabilises the polyacrylamide which, below 5% or so, would become too soft to handle.
doi:10.1007/978-1-4939-8793-1\_18 and doi:10.1006/abio.1995.1004 may be useful.
I find tank blotters more reliable especially with large proteins than semidry.
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