In vitro Methylatietest Assay om Protein Arginine methylatie Bestudeer

The overall goal of this procedure is to establish the methylation status of protein arginine methyl transferase or PRMT substrates. This is accomplished by first expressing epitope tagged PRTs in mammalian cells. The P RMTs are then immuno precipitated from the cell lysates using anti hemagglutinin or anti HA coated SRO speeds, followed by an extensive wash to remove non-specifically bound proteins.

Next, the washed beads are incubated with the substrate protein purified from bacteria and tritium labeled snail L methionine, or sam. Finally, the reaction is stopped and the methylation status of the substrate is determined via sodium syl sulfate, poly acrylamide gel electrophoresis or SDS page analysis followed by auto radiography. Ultimately, the ability of p RMTs to methylate the substrate is determined by comparing the methylation reactions using immunoprecipitation performed on the lysates of empty vector and BRMT transfected cells.

Proteomic approaches or immuno blotting with methyl arginine specific antibodies can sometimes be misleading, often providing false postal results. Most importantly, they cannot provide a direct evidence in support of PRMT substrate specificity. The main advantage of the in vitro methylation assay that I will be demonstrating in this video is that it is sensitive and consistently reveals if the identified proteins where PRMT substrates On the day before transfection seed 750, 000 human bronchial epithelial cells in 10 milliliters of culture medium.

In a 100 millimeter culture dish, swirl the dish to evenly disperse the cells and incubate at 37 degrees Celsius in a 5%humidified CO2 incubator. The next day aspirate the medium and add five milliliters of culture medium without serum and antibiotics swirl and aspirate the medium. Repeat this step one more time and finally add five milliliters of serum antibiotic-free medium, and proceed to transfection.

To perform the transfection first dilute six micrograms of PRMT plasmid DNA into 750 microliters of serum free medium in a sterile 1.5 milliliter plastic centrifuge tube in another 1.5 milliliter plastic centrifuge tube. Dilute 30 microliters of transfection reagent into 750 microliters of serum free medium. Then transfer the diluted transfection reagent solution to the tube containing diluted DNA mixed gently by pipetting up and down several times.

Incubate the transfection mixture at room temperature for 15 minutes. Next, pipette the transfection mixture gently onto the cells and swirl to mix evenly. After a three hour incubation at 37 degrees Celsius, aspirate the medium containing the transfection mixtures from the cells.

Then add fresh cell culture medium supplemented with serum and antibiotics. Following a 48 hour incubation at 37 degrees Celsius lyce cells in one milliliter of lysis buffer. Collect the lysates onto a plastic centrifuge tube with a cell lifter.

Then incubate the lysates on ice for 30 minutes. Next, centrifuge. The lysates at 15, 000 GS for 10 minutes on a benchtop centrifuge.

To clear the lysates from cell debris for purification of P mts. Incubate three milligrams of cell lysates with 30 microliters of a 50 to 50 suspension of anti HA affinity matrix in phosphate buffered saline. Or PBS rotate overnight at four degrees Celsius in a tube rotator.

The next day, spin the tubes at 14, 000 GS on a benchtop centrifuge for two minutes to collect the beads. After discarding the snat, wash the beads with one milliliter of RIPA buffer followed by centrifugation as before. After washing the beads three times with RIPA buffer, wash the beads once with 100 microliters of methylation buffer.

After the centrifugation, remove the supernatant. The beads containing the immobilized p RMTs are ready to be used in the methylation assay. Perform the methylation assay in a separate area designated for radioisotope.

Work in compliance with the institution's protocols for working with radioactive isotopes. Prepare purified glutathione, s transferase, or GST substrate as described in the text protocol. Combine two micrograms with a master mix containing one x methylation buffer and micro curie of tridium labeled snail L methionine.

Add the mixture to the immobilized p RMTs and incubate at 30 degrees Celsius for one hour. After one hour, stop the reaction by adding 10 microliters of two XSDS. Sample buffer following denaturing of the samples at 95 degrees Celsius for 10 minutes.

Separate the samples by SDS page later. Transfer the separated proteins from the SDS gel onto a nitrocellulose membrane by using a semi-dry electrophoretic transfer. After the transfer, cover the membrane with an auto radiographic image.

Intensifier and gently rock for two hours at room temperature After two hours, pour off the intensifier and retain for later use. Quickly air dry the membrane on a filter paper, seal it in a plastic bag, and expose the membrane to an x-ray film at minus 20 degrees Celsius. Sufficient exposure times range between five and 30 days.

After achieving sufficient exposures, transfer the membrane to a plastic box containing blocking buffer and gently rock for one hour. Discard the blocking buffer as radioactive waste. Then incubate the membrane with anti HA antibody in T tris, buffered saline with tween 20, abbreviated TBST overnight at four degrees Celsius with gentle rocking the next day.

Wash the membrane with TBST three times. Then incubate the membrane with horse radish peroxidase labeled anti mouse secondary antibody in TBST at room temperature for one hour with gentle rocking. After washing the membrane with TBST three times, perform chemiluminescence detection of proteins with horseradish peroxidase substrate after detection of HA tag P RMTs.

Incubate the membrane with poncho S staining solution for five minutes. At room temperature, quickly wash the membrane three times with water before scanning the image. To detect the GST tagged substrates, the abilities of HA tag PMT one to methylate GST tag G three PP one were determined by an in vitro methylation assay.

HA TAG prm T one purified from B2B cell. Lysates was employed in a methylation reaction containing GSTG three, BP one and one micro curry of tritium labeled snail L methionine. As a methyl donor, p MT one could efficiently catalyze the methylation of GS TG three BP one methylation reactions using pull downs performed on lysates of empty vector.

Transfected B2B cells served as a negative control. Immuno blotting with anti HA antibodies confirmed the expression of p rmt. One ponchos staining was then used to demonstrate equal loading of GSTG three BP one in both the reactions.

After watching this video, you should have a good understanding of how to perform an in vitro methylation assay.