We already have in place HPLC/UPLC based methods, but these are ' stopped time' based, so tedious for kinetic studies. Looking for a colorimetric assay (eg. enzyme coupled?) that would target the co-product, S-adenosylhomocysteine. Any thoughts appreciated.
Do you know this paper?
Analytical Biochemistry, Volume 326, Issue 1, 1 March 2004, Pages 100–105
An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases
Cheryl L Hendricksa, Jeannine R Rossb, Eran Picherskyc, Joseph P Noelb,
Zhaohui Sunny Zhou
http://www.sciencedirect.com/science/article/pii/S0003269703008236
There is a kit SAM methyltransferase kit available in G Bio sciences which can serve to your purpose.
Hi,
I did enzymatic ELISA tests on methyltransferases during my first year of master. I send you the report in attachment, hoping that this can give you an idea to solve your problem. As you can see the method seems to be suitable for kinetic studies too.
Silvia
you may try to measure released S-Methyl-cysteine. I developed a GC-MS assay which I attach.
sorry, the message got broken when I attached the article. I measured MeSCy in human urine, which is a dirty matrix, I don't know whether you have a cleaner matrix, which makes measurement simpler. You may try for an alternative internal standard (deuterated D3C-SCy is not commercial, I think: we prepared but now we do not have any more) such as nor-valine. Hope to be useful, all luch and best regards, FMRubino
If you have a good antibody, AlphaLISA works very well in epigenetic assays.
http://www.perkinelmer.ca/en-ca/Catalog/Category.aspx?ID=AlphaLISA%20for%20Epigenetics%20Research&M=BIO
The simplest method I know of is to detect the free thiol of the product S-adenosylhomocysteine (SAH) using the fluorogenic reagent Thio-Glo-1. It does not require any coupling enzymes and has sensitivity for less than 1 micromolar. Naturally, you can not have any other thiols present, such as dithiothreitol. The assay can be done in a very small volume using a fluorescence plate reader, and the reaction can be followed continuously.
Correction to the above: A coupling enzyme is required. It is S-adenosylhomocysteine hydrolase (SahH). It can be cloned from the genome of the bacterium Pseudomonas aeruginosa and expressed in E. coli for purification.
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