I wanted to do negative staining of polyacrylamide gel, using zinc sulphate or zinc acetate salts. The procedure I followed was 5 minutes incubation with 1% sodium carbonate, followed by a long incubation of 30 min in 200 mM imidazole made in 0.1% SDS after which I incubated the gels in 0.1 M zinc sulphate and also tried with zinc acetate. no staining could be observed even in 5 minutes or 10 minutes whereas the protocol says it should have been stained in few seconds. what could be the possible reasons? Can anyone please give me a tried and tested protocol and also explain me the reasons behind using them? Zinc acetate made a very opaque solution, should it be dissolved in ethanol?
Hi Mansi,
perhaps this link helps a bit. It states 10-15 minutes.
http://www.gbiosciences.com/ReversibleZincStain-desc.aspx
Perhaps the following article also helps:
Chen HM Revisit of imidazole-zinc reverse stain for protein polyacrylamide gel electrophoresis.
Methods Mol Biol. 2012;869:487-95. doi: 10.1007/978-1-61779-821-4_43
hi Pajkos
the g-biosciences have provided the solution and i tried doing it making my own, i would like to know why it did not.
the other article i have to see, its not available free online.
Others have sent similar, but there is a protocol in Current Protocols in Protein Science (2007) "page" 10.6.3 2007. It uses Zinc sulphate & imidazole to stain. I have used this and can see the negative bands where a lot of protein is loaded. It is considered reversible because the white precipitate can be removed using 0.3% citric acid. However, I have not tried that part.
hey thanks so much Stacy but it would be so kind of you if you could send me the pdf i'm unable to access it.
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