Hello,
I have read on Thermo Fisher's website that EMSA can be done without any kind of labelling, just by post-staining the gel with a fluorescent DNA dye. However, information about this is very scarce and publications using this method are hard to find.
It seems 10x easier without labelling, then why are so few people doing it ?
Do you have any experience with EMSA using fluorescent dye or ethidium bromide vs conventionnal labelling ?
FYI, I intend to check protein binding to complementary oligonucleotides of 30-100bp, using purified protein extracts, so I am not too concerned about non-specific binding.
I have done EMSA with ethBR. Do you have any specific question or just want a general protocol?
I am just wondering if you can get good results (crisp bands with low background/smear) with EtBr or fluorescent dies, since I can hardly find anything in the litterature about this way of doing EMSA.
Yes you can. The reason why you do not see it so much in publications has to do with the complex size (agarose EMSA with EthBR) is usually used for larger protein complexes therefore you might not see the shift if your complex size is too small that why people tend to go for polyacrylamide gel electrophoresis (and also people want to use a low amount of the probe which is not possible with conventional gels unless you use more sensitive dye like syber green). However, it can be used for your system since it is protein extracts. I would recommend that you do not cast EthBR in the gel but only stain afterwards as casting it in the gel might affect your complex migration.
Best of luck!
Hanna is right. Yes you can. But flourescent-dye labeling is more frequently reported. See the link: https://www.ncbi.nlm.nih.gov/pubmed/27929467. Fluorescent probes can be detected in-gel with the aid of an appropriate imaging system, but this method has not been very popular to date because expensive instruments are required and their sensitivity does not yet compare to radioactive probes.
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