The goal of this procedure is to measure the enzymatic activity of individual members of Protein Arginine Methyltransferase family in cells. The activity is measured by determining their arginine methylation levels and total levels of bio-marker protein using quantitative fluorescent western blotting. The advantage of the described method is the straightforward performance in any lab with cell culture and fluorescent Western blot capabilities.
The detailed guidelines regarding bio-markers, antibodies and inhibitor two compounds are provided in the article. And here we described crucial steps involved in the experimental procedure. And the procedure is shown for 24 well format.
First, prepare lysis buffer, add benzonase and protein inhibitor cocktail freshly before use. Remove all media from wells. Wash with 100 microliter PBS, and add 60 microliters of lysis buffer to the well.
Incubate for one minute at room temperature, rocking the plates to distribute the lysis buffer over the cells. Then add three microliters of 20%SDS to find a 1%concentration and mix by gentle shaking. Transfer lysate into Eppendorf tubes and keep it on ice.
Determine protein concentration of your samples using BCA protein assay kit, or use any other methods which tolerates 1%SDS in solution. After adjusting the protein concentration with lysis buffer to be equal across the samples at 20 microliters of four times loading buffer to 60 microliters of cell lysate, and heat at 95 degrees for five minutes. The addition of benzonase to lysis buffer rapidly hydrolyzes nucleic acids, which reduce cell lysate viscosity.
Lysates with benzonase are not viscous. Lysates without benzonase have a big glob of gooey DNA that will not pellet when spun. Load to five to 20 micrograms of total cell lysate for histone proteins, and 20 to 100 micrograms for other proteins in a gradient four to 12 bis-tris protein gel.
Around the jelly in mops as the surrounding buffer for about two hours at 100 volts, or until the blue loading dye reaches the bottom of the gel. If you perform a wet transfer, assemble the transfer sandwich in ice cold tris-glycine transfer buffer. Activate PVDF membrane by soaking in methanol and equilibrate gel in transfer buffer for 30 seconds prior assembly.
Place sponges filter paper, PVDF membrane and gel according to manufacturer's instructions. Transfer proteins in tris-glycine transfer buffer at 70 volts for a one to one and a half hours on ice. After transfer, incubate the membrane for 30 minutes in blocking buffer.
Rinse with wash buffer, and incubate with primary antibodies overnight at four degrees. Next day, wash membrane three times for five minutes with wash buffer, incubate with fluorescently labeled secondary antibodies for 30 minutes at room temperature, and wash three times for five minutes with wash buffer. Place the membrane face down on the glass plate of the fluorescent Western blot imager.
Cover with the rubber sheet and remove any air bubbles. Read the signal at 700 and 800 nanometers. Once the scan is done, go to image and adjust 700 and 800 channel signal to see bands clearly.
Here the 700 channel signal shown as red represents total protein levels, and 800 channel signal shown as green represents the levels of methylated protein. Next, go to analysis and select median top and bottom reading. To measure bands intensity, select draw rectangle tool and draw the shape around the bands you want to quantify.
Go to shapes where you find intensities of 700 and 800 channels. The column called signal provides intensity levels with background subtracted. The results can be copied directly to the Excel file.
Here is the example showing the analysis of PRMT1 activity in cells upon inhibition with PRMT type one inhibitor MS023. PRMT1 activity is measured by determining the histone H4 arginine 3 asymmetric dimethylation levels. Here, the methylation levels, green bands go down in a dose dependent manner, while the total histone levels red bands remain unchanged.
To determine compound IC50, plot the logarithm of inhibitor concentration against histone H4 arginine-3 asymmetric dimethylation levels normalized to total histone levels followed by non-linear fit analysis. The described method allows for a liable and reproducible double detection of protein methyltransferase's activity in cells. The advantage of the method is that it utilizes well known Western blotting methodology that is accessible for academic labs and can be easily modified depending on the lab equipment.
The assay details including bio-marker proteins, antibodies and chemical two compounds for each individual PRMT family member are provided in the article.
