Silver: Methylation Assay
The Enzymes: The Prmt enzymes can be purified from many sources: by immunoprecipitation from mammalian cells, by GST or His tag purification from bacteria or insect cells.
Note: Prmt5 seems to require a cofactor or a post-translational modification to be fully active. As a consequence, Prmt5 expressed and purified from bacteria is virtually inactive.
The Substrates: The substrates can be purified from the same sources as described above, although they may already be partially methylated in mammalian cells, which in turn reduces their ability to be further methylated in vitro. Treating the cells with 20 uM AdOX for 72h before purification should help minimize this problem.
MBP (Myelin Basic Protein) works well as a substrate for Prmt5 and can be purchased from Upstate Biotechnologies (cat# 13-110).
Numerous RGG-domain-GST fusion substrates have been published and should be freely available to the scientific community. Our lab has the RGG domain of Npl3 and the bacterial expression vector for ti is available upon request.
Note: If you are planning to purifiy either the substrate or the enzyme from mammalian cells through Flag purification, beware: the Flag antibody immunoprecipitates very well the endogenous Prmt5 as a contaminant, even using stringent IP conditions (500mM NaCl does help reduce but does not eliminate the co-purification of endogenous Prmt5). So you’ll absolutely need to include a mock IP as control (Flag IP-ing a cell extract prepared from a control cell line that does not express a Flag-tagged protein).
Curious observation: Purifying Flag tagged-GFP from 293 cells (as a negative control) does not co-precipitate endogenous Prmt5 while copious amount are copurified along with many other Flag-tagged proteins.
The Methyltransferase reaction: A common reaction buffer is:
- 150 mM NaCl
- 20 mM Tris pH 7.5
- 1 mM EDTA
- 0.02% Triton
- Your favorite protease inhibitors
The reaction requires 50 to 500ng of enzyme, 1 to 5 ug of substrate, and 0.5 ul of 3H-SAM (tritiated Adenosyl-L-methionine, S-[methyl-3H] 55-85 Ci/mmol, NEN cat# NET155H), for a final reaction volume of 10 to 25 ul. They are incubated at 37ºC for 3h to overnight.
You’re almost home: At this point, the reaction can be stored in the freezer, or further processed through polyacrylamide gel electrophoresis, staining, fluorography, drying. Well, you’re not home quite yet, so freeze it...
PAGE separation and staining: The reactions are resolved through classic denaturing polyacrylamide gel electrophoresis. NuPAGE 4-12% Bis-Tris gradient gels (Invitrogen cat# NP0329BOX) will give you optimal sample separation, but are a bit expensive. Following electrophoresis, stain the gel with Coomassie, and then destain, following any age old protocol.
Note I: Performed the staining procedure on a roking platform placed in a fume hood. The classic Commassie receipe contains Methanol, a very toxic chemical.
Note II: do not stain using a Silver stain procedure, as the tiny silver ions around the protein will block the weak alpha emmision of the triated substrate.
Fluorography: After destain, the gel is incubated on a shaking platform for 30 min at room temperature in a nasty-smelling solution like EN3HANCE™ Autoradiography Enhancer (Perkin Elmer cat#6NE9701), and the fluor is then precipitated by incubating the gel in water for 30 min to 1h. The gel will turn white as soon as it is palced in water. This is normal!
Note: Use a glass container for these steps as the Enhance, in addition to smelling bad, will ruin a plastic container. Do not let the gel in water for much longer as the bands will become more and more diffuse, and you’ll loose resolution and sensitivity.
After this step, insert an appropriately cut sheet protector under the gel and lift it out of the tray. Place 3 sheets of Whatmann paper on top of the gel and lgihtly press using a flat surface such that the gel sticks to the paper. Flip the whole stack, and carefully peel away the sheet protector (the gel should remain stuck to the Whatmann paper). Cover with Saran wrap and dry fro 1h , according to your gel dryer caracterisitcs.
Exposure: Peel away the Saran wrap from the dried gel, and expose to film. Kodak “Biomax” MS film in combination with BioMax TranScreen LE Intensifying Screen (Fisher cat# 05-728-67) seem to work well for this application.
Exposure time will vary, from a few hours to sevral days (up to a month), so go home now…