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In-Vitro protein methylation assay using 3H-SAM

Author: Levy Lab, updated date: , view: 65, Q&A: 0
Tags: In vitro and Protein Methylation

Procedure

Dan Levy's Lab 2018

Lital Weil

In-Vitro protein methylation assay using 3H-SAM

  1. Purify recombinant proteins followed by dialysis against PBS buffer pH 7.4, 10% glycerol.

Note:
a. Try to avoid the use of buffers containing reducing agents and metal chelators as these might have an inhibitory effect on the enzymatic reaction.
b. Be cautious! From this point on, reagents are radioactive and must be handled with appropriate caution.

Methylation reaction

  1. Set up a 1.5 mL microcentrifuge tube on ice for each reaction assay.

  2. In each tube, mix: 1ug of recombinant methyltransferase, 5ul of 5×PKMT buffer (10 mM Tris–HCl (pH 8), 2% glycerol, 0.8 mM KCl, 1 mM MgCl2), 13uM S-adenosyl-L-[methyl-3H]-methionine (3H-labeled SAM) (AdoMet; PerkinElmer), 1-3ug of the desired substrates and H2O (sigma water, Molecular Biology Reagent) to a final volume of 25ul.

Note: Be sure to use enough protein to allow detection by SDS-PAGE followed by Coomassie staining.

  1. Incubate the reaction tubes at 30°C for 2-18h.

  2. The reaction is stopped by adding 10ul of 5×protein sample buffer (250mMTrisHCl (pH 6.8),10% SDS, 30% glycerol, 5% β-mercaptoethanol, bromophenol blue) followed by boiling the samples at 95°C for 5min.

Analysis

  1. For Coomassie and autoradiogram analysis, run the samples on two different SDS-PAGE gels.
    Note:
    a. Be caution! The running buffer in the gels running tank might be contaminated with free S-adenosyl-L-[methyl-3H]-methionine.
    b. Take into consideration the molecular weight and the concentration of the proteins as well as the methyltransferase activity, and run gels accordingly.

  2. Stain the gel in Coomassie Blue stain for 20min or over-night (Expedeon, InstantBlueTM).

  3. For autoradiogram, transfer samples from one gel to polyvinylidene difluoride (PVDF) membrane. Allow the membrane to dry completely, for at least 20 min, then mark the protein scale marker at the relevant size marks, using 1:400 diluted SAM. Again, allow the membrane to dry completely, for at least 20min.

  4. Expose the membrane to a Fujifilm imaging plate, then set the plate at the appropriate cassette for 2-18h.

  5. Read the radioactive signal using Typhoon™ FLA 7000 biomolecular imager.



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