Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

The small Rho GTPase is a protein of a superfamily with diverse cellular function. For example, these proteins are involved in the regulation of cell fate, cellular response to a stress, cellular motility, and cell cycle. The activity of a small Rho GTPase protein are regulated by post-translational modification.

These modifications are involved in tight regulation of their activity. As an example, protein prenylation and GTP bond are the most important post-translation modification of these proteins. My lab has recently modified and developed simple method to investigate the post-translational modification of these superfamily proteins.

For this purpose, we have used 251 glioblastoma cell lines, and targeting them with simvastatin, and measured post-translational modification of these proteins. The method involves sub-cellular fractionation and GTP bound protein to this protein superfamily. For this purpose, we have used localization of RhoA, and GTP bound to RhoA to measure the post-translational modification of this protein.

Remove cells from the 37 degree incubator. Look at the cells under the microscope to confirm confluency. The cells should be at 70 to 80 percent confluent.

Wash the cells once with cold PBS. Add five milliliters of EDTA, and place the cells back into the 37 degree incubator for five minutes. After five minutes of incubation, collect EDTA with cells into a 15 mil tube.

Place the tube in an ice box, and proceed to the centrifuge. Set up the centrifuge to 1500g at four degrees, and spin the cells for five minutes. Following the spin, remove the supernate, and add one milliliter of cold PBS.

Triturate the cells well. Transfer the cell mixture to a new labeled 1.5 milliliter tube. Following transfer, proceed to the centrifuge.

Set the centrifuge to 1500g at four degrees, and spin the cells for five minutes. Check the pellet size. Place the samples on ice.

Discard the supernate completely, without disturbing the pellet. Add Buffer One. Mix well by pipetting up and down.

Proceed to sonication. Set the sonicator for five cycles, five seconds each, and repeat three times. Sonication should proceed on ice, but is not shown in the video, for illustrative purposes.

Proceed to the ultracentrifuge. Use an ultracentrifuge to separate the cell homogenates into cytoplasmic and membrane fractions. Set the centrifuge to 100, 000g for 35 minutes, at four degrees.

Check the pellet size. Separate the supernate. Collect the supernate completely, being careful to not disturb the pellet.

The supernate is the cytosolic fraction. Place the supernate in a newly labeled tube. Add Buffer Two to the pellet.

This is the membrane fraction. Mix well by pipetting up and down. Proceed to protein determination and Western blot sample preparation.

Load the samples on to an SDS-PAGE using a 15%gel. Confirm protein transfer by visualizing the molecular weight marker, or using a Ponceau stain. Add primary antibodies for immunoblotting analysis.

We use Rac1/2/3, Cdc42, and RhoA. Bring the culture plates out of the incubator. Look at the cells under the microscope to confirm confluency.

The cells should be 70 to 80%confluent. Place the culture plate on ice. Aspirate the media and wash the cells with ice cold PBS pH'd to 7.2.

Aspirate the PBS. Tilt the plates on ice for an additional minute to remove all remnants of PBS. Residual PBS adversely affects this assay.

Lyse the cells in an appropriate volume of ice cold lysis buffer containing protease and phosphatase inhibitors. Harvest cell lysate with the cell scraper. Incline the culture plate for this technique.

Transfer the lysate into a labeled, ice-cold cryo tube, and keep on ice. Mix thoroughly using a vortex. Keep 10 microliters of the lysate for a protein assay.

Snap freeze the remaining cell lysate in liquid nitrogen. Transfer the snap frozen cryotubes to a minus 80 freezer. Store these samples.

Note, samples should not be stored longer than 14 days. Prepare cell lysates from the other culture plates by repeating steps one to eight, one by one. Work quickly, and never leave the samples on ice for longer than 10 minutes.

Never handle all culture plates simultaneously. Measure the protein concentration. Calculate the volume of lysis buffer to normalize the concentration of protein between the samples.

Note:one milligram per milliliter is usually the best, however, 0.3 to two milligrams per milliliter can be detectable. Prepare a blank control by adding 60 microliters of lysis buffer, and 60 microliters of binding buffer to a microtube. Prepare a positive control by adding 12 microliters of Rho control protein, 48 microliters of lysis buffer, and 60 microliters of binding buffer.

Take the Rho affinity plate out of its bag and place on ice. Dissolve the powder in the wells with 100 microliters of ice cold distilled water. Keep the plate on ice.

Thaw the snap frozen cell lysates in a water bath set to 25 degrees Celsius. Add the calculated volume of ice cold lysis buffer to each sample to normalize the protein concentration. Transfer 60 microliters of normalized ice cold samples to microtubes, and add 60 microliters of binding buffer.

Mix thoroughly, and keep on ice. Completely remove the water from the microplate by vigorous flicking, followed by five to seven hard taps on a lab mat. Add 50 microliters of the normalized samples, a blank control, and a positive control to the wells in duplicate.

Place the plate on an orbital shaker for 30 minutes at four degrees Celsius. Note:the speed of the shaker should be set to 300 rpm. Clear the samples from the plate by flicking, and wash twice with 200 microliters of room temperature washing buffer.

Vigorously remove the washing buffer from the wells after each wash by flicking, followed by tapping, and keep the plate on the bench at room temperature. Add 200 microliters of room temperature antigen-presenting buffer into each well, and incubate at room temperature for two minutes. Flick out the solution from the wells, and wash three times with 200 microliters of room temperature washing buffer.

Add 50 microliters of freshly prepared one to 250 anti-RhoA primary antibody to each well. Place the plate on an orbital shaker for 45 minutes at 300 rpm, set to 25 degrees Celsius. Flick out the solution from the wells, and wash three times with 200 microliters of washing buffer at room temperature.

Add 50 microliters of freshly prepared one to 250 anti-RhoA secondary antibody to each well. Place the plate on top of an orbital shaker for 45 minutes, at 300 rpm, set to 25 degrees Celsius. Prepare the HRP detection reagent by mixing equal volumes of Reagent A and Reagent B.Flick out the solution from each well, and wash three times with 200 microliters of room temperature washing buffer.

Add 50 microliters of freshly prepared HRP detection reagent to each well. Read the luminescence signal within three to five minutes to obtain a maximal signal. Analyze the results using an appropriate software package.

Our results show that simvastatin decreased the localization of RhoA in the membrane fraction. What increased is accumulation in the cytosolic fraction. Interestingly, simvastatin increased GTP bound to the RhoA.Why?

We expect simvastatin decrease this activity. It is very important they measure both sub-cellular localization of this protein, and measure GTP binding to this protein to have a final evaluation of the activity of the small Rho GTPases.