In vitro Methylation Assay
- Isotope Used: [3H]. 2 μCi per labeling reaction.
- Before proceeding, wear labcoat, double gloves, protective goggles, and dosimeter (whole body badge and ring) on your labcoat.
- Place absorbent diaper in work area to contain possible spills.
- Always change if gloves become wet.
- Use filtered pipette tips to ensure no contamination of pipetteman.
- Discard all radioactive wastes in designated [3H] shielded radioactive waste container (liquid or dry waste.
- Plastic Tubes
- Pipette Tips
- Absorbent Pads
- SDS Acryl/Bis Gels
- SDS Buffer
- Coomassie Buffer
- Coomassie Destain Buffer
- 50 μL reaction set-up: 5 μL 10X PBS Solution (Make a Master Mix including [3H] SAM), 2 μCi [methyl 3H]-SAM
(78 Ci/mmole), 3 μg Enzyme, 3 μg Substrate, Q to 50 μL with H2O. Add enzyme and substrate first, and then add SAM
to PBS Master Mix. Pipette Master Mix well.
- Substrate Panel:
- RG Peptide
- Negative Control
- No Enzyme
- Positive Control
- PRMT7 (Signal after 1 week x-ray film in -80°C Freezer)
- Substrate Panel:
- Incubate reaction on desktop overnight with timer. Record temperature of room and amount of time of reaction.
- Add 25 μL 4X SDS Dye, total 75 μL reaction with 2 μCi SAM.
- Boil samples 5 minutes at 95°C.
- Run 10 to 15 μL of reaction on 10% to 15% Acryl/Bis Gel. Use 15% gel if using Histones as a substrate.
- Run gel until dye front of sample is still on gel, few mm from bottom of gel, [3H] SAM still in gel.
- Check contamination of SDS Buffer by Scintillation Counter.
- If Buffer is contaminated, aspirate liquid waste (approximately 500 mL) from electrophoresis into liquid [3H] waste container.
- Cut off bottom of gel where dye front / [3H] SAM is and dispose in solid [3H] waste container.
- Decontaminate gel plates and place gel in box for Coomassie Stain for few hours.
- Dispose of Coomassie Stain in liquid [3H] waste container.
- Add Destain Solution and rock overnight.
- Pour Destain Solution into liquid [3H] waste container and add more, rocking until gel is destained well. All of [3H] SAM should be contained within the Coomassie Stain or Destain Buffer and disposed of at this point.
- Take picture of gel on densitometer.
- Add ENHANCE to gel, rock, and pour enhance into specific container for waste.
- Add H2O to gel, dry gel on Gel Dryer, then expose on gel.
∗∗All liquid waste generated would be tested for presence of radioactivity and being disposed of as regular chemical waste if none are found.
∗∗Perform swipe test on benchtops, centrifuges, gel plates and apparatus, and gel dryer after completion of experiment and perform scintillation counting to ensure no contamination.
IF CONTAMINATION IS DETECTED, CLEAN THE WORK AREA AND RE-SURVEY WITH SWIPE TEST.
Download Protocol: In vitro Methylation Assay
(note requires Microsoft Word to open)