The overall goal of this procedure is to follow activation of RO a specific G-D-P-G-T-P exchange factors or gafs in cultured cells by making use of a mutant RO A GST fusion protein that has high affinity for activated gs. This is accomplished by first producing the GST tagged mutant row protein in bacteria. Next the bead coupled GST row A is purified.
Then the purity and concentration of the GST RO A protein is assessed using SDS page. Finally, cell lysate are prepared and a GEF precipitation assay is performed with the GST RO A protein. Ultimately, results can be obtained that show increased precipitation of activated GS in treated cells through western blot analysis of the precipitated proteins with a specific anti GF antibody and quantification by dense cytometry.
This method can help answer a number of exciting questions in the raw GTPS field, such as how are the many row G-D-P-G-T-P exchange factors regulated in a signal specific manner and how is their activity coordinated? Generally individuals new to this method will struggle initially because the GST RO G 17 A protein is relatively unstable and can break down during preparation. It is also important to remember to perform the GF precipitation assay quickly and consistently Demonstrating this procedure will be Pfizer Vahid, a master student from my lab To transform e coli.
Quickly tho an quat of DH five alpha competent cells in an ice bath add one microliter of 25 to 50 nanograms per microliter PX row A G 17 A DNA. Flick the tube to mix and incubate on ice for 30 minutes. Heat chuck the cells at 42 degrees Celsius for 45 seconds and placed back on ice for two minutes.
Add 900 microliters of sock medium and grow for one hour at 37 degrees Celsius with shaking. Using a bent sterile pasta pipette spread 50 to 100 microliters of the transformed bacteria on a previously prepared LB agar and persin plate. Incubate the plate right side up in a 37 degrees Celsius incubator for five minutes and then invert and grow overnight.
Pick a well isolated colony of transformed bacteria and use it to inoculate 50 milliliters of LB with ampicillin. Grow overnight at 37 degrees SIUs with agitation When the culture has reached full density, dilute it with 450 milliliters of LB ampicillin and grow it for an additional 30 minutes. At 37 degrees Celsius, induce the bacteria to produce row protein by adding 500 microliters of 100 millimolar IPTG to a 500 milliliter culture.
Reduce the temperature to 22 to 24 degrees Celsius and grow for around 16 hours. Spin the culture at 3, 600 times G for 10 minutes at four degrees Celsius. Freeze the pellets for at least one hour or preferably overnight at minus 80 degrees Celsius.
After preparing 200 milliliters of lysis buffer as outlined in the written protocol, make lysis buffer plus by supplementing 10 milliliters with one millimolar DTT one millimolar PMSF and one protease inhibitor tablet working on ice. Add 10 milliliters of lysis buffer plus to the Thor pellets and spend thoroughly by pipetting and vortexing. Sonicate the suspension on ice for one minute at setting four with 50%pulse.
Spin the sonicate lysate 15, 000 to 20, 000 times G for 15 minutes of four degrees Celsius and transfer the clarified sonicate to a sterile capped 15 milliliter tube. Prepare the glutathione sphero beads by gently mixing the 75%slurry and using a wide boar tip to transfer 335 microliters into a 15 milliliter tube. Add 10 milliliters of cold PBS and spin it 500 times G for five minutes at four degrees Celsius.
Discard the suen agent. Add one milliliter of lysis buffer plus to the beads and spin again. Discard the supinate again and add lysis buffer.
Plus, to make a 50%slurry, combine two 50 microliters of equilibrated bead slurry with the prepared supernatant. Rotate at four degrees Celsius for 45 minutes. Prepare HBS plus by supplementing 100 milliliters of HBS buffer with five millimolar magnesium chloride and one millimolar DTT centrifuge the supinate and beads at 500 times G for five minutes of four degrees Celsius.
Discard the supinate and wash the beads twice with 10 milliliters of lysis buffer plus and twice with 10 milliliters of HBS plus. After the final wash, make a 50%slurry by resus suspending the beads in HBS plus supplemented with protease inhibitor. Dilute 10 microliters of the final bead preparation with two times lam ly sample buffer containing beamr cap two ethanol.
Then make three BSA standard samples by mixing 10 microliters. Five microliters and then 2.5 microliters of a two milligram per milliliter. BSA stock with 10 microliters of lammy buffer.
Boil the samples for five minutes, then briefly spin and run them all with a molecular weight marker on a 10%SDS poly acrylamide gel after staining and des the gel estimate the GST row A G 17 A using the BSA standards as a reference. Aliquot and equal volume of beads containing around 10 to 15 micrograms of protein into 1.5 milliliter micro centrifuge tubes. To prepare for the GF pull down assay, grow the cells to be used to confluence in 10 centimeter dishes and serum deprive them for at least three hours before the assay.
Just before the start of the assay. Prepare lysis buffer plus with protease inhibitors working on ice. Remove culture medium from the dishes and wash with ice cold HBS.
Remove all the HBS and add 700 microliters of lysis. Buffer plus to each dish. Swell the plates to cover all areas.
Then scrape and collect lysates into numbered 1.5 milliliter tubes. Spin at 15, 000 times G for one minute at four degrees Celsius. Save the supinate for the supernatant for the assay if the cell number was equivalent in each plate, remove 30 of each supernatant and mix with 30 microliters of two times reducing laly sample buffer.
Then boil and set aside on ice. Add the remaining supernatants to aliquots of the GST row a G 17 A beads and rotate for 45 minutes at four degrees Celsius. Spin the beads at 6, 800 times G for 10 seconds at four degrees Celsius.
Discard the supinate and wash the beads three times with lysis buffer. Using a one CC syringe fitted with a 30 gauge needle completely remove the final wash and add 20 microliters of two times reducing laly sample buffer. Boil the samples for five minutes, spin to pellet the beads and run the total cell lysates and precipitated protein samples on the appropriate percentage of SDS page gel for the size of the GF being studied.
Finally, perform western blotting to detect the relevant GFA successful GF assay. Detecting activation of the exchange factor GFH one is shown here. The row A G 17 A protein captures some GFH one from the control cell lysates suggesting that G FH one has basal activity.
The amount precipitated however increased in cells treated with the inflammatory cytokine tumor necrosis factor alpha consistent with the notion that TNF alpha activates G FH one. Importantly, the total cell lysate shows similar amounts of gfh one in the control and the treated sample, suggesting that the treatment did not alter G FH one levels and that the input used in the assay was equal. While adapting this, this procedure, it's important to maintain the GSE RO G 17 A protein and the CELLIS eights on ice.
Perform the GF assay quickly and consistently, and to use adequate positive controls that will enable invalidation and troubleshooting.
