I'm trying to measure the activity of a methyltransferase enzyme for histones and would like to be able to quantify the results. I have tried using 3H labeled SAM and measuring the activity by scintillation counting, but the counts are usually very low and only 2-3 fold above background. I could potentially use a film and leave it for several days to increase the signal, but would like to try a faster method or a non-radioactive one. We need to be able to collect signal from mono- di- and trimethylatiion and don't have the antibodies available for all forms to do wb. Also,the amount of substrate that I have is very limited.What is the best way to do this?
Hi Florence,
The technique to be used largely depends on - what context you wish to measure the histone methylation increase or decrease, are you looking at global change in histone methylation or at a specific region of DNA. I think the most reliable technique is western Blot, which will enable you to between your treatment or control, the change in histone methylation level. And for looking at changes in histone methylation at specific regions would be IP followed by WB.
Regards,
Prasad.
Hi Florence,
A large number of histone modifications (59 to be precise, with many lysine and arginine methylation states) can be identified using the Active Motif Histone Peptide Array: http://www.activemotif.com/catalog/668/modified-histone-peptide-array
It's not the cheapest option and I don't know if you have sufficient material to do the experiment. It's by far the most comprehensive tool I'm aware of though.
There are some colorimetric assays for global methylation available, for example: http://www.epigentek.com/catalog/epiquik-dnmt-activityinhibition-assay-ultra-kit-colorimetric-p-2823.html
I doubt that they are as quantitive as H3 but one could check them. :)
Alternative is a mass-spectrometry of Arginines, me-R forms. I am sure it's could be done only through outsourcing.
For global methylationanalysis you can use western blot with specific antibodies against the different methylated lysine positions and methylation status (i.e. mono di and tri). For loci-specific analysis you could use similar antibodies in chromatin immunoprecipitation assays
A bit of clarification here: I'm doing an in vitro enzyme assay, so I have purified the methyltransferase I'm working with and have the histone substrates and I measure the activity by using 3H-labeled SAM and scintillation counting, but the counts are very low and doing wb is not an option either since antibodies are not available. What other method could I possibly use?
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