The overall goal of the following experiment is to label the proteins of interest with radioactive phosphates in order to quantify the proteins, cellular phosphorylation levels. This is achieved by culturing cells in the presence of phosphorus 32 to radioactively labeled the phosphate modifications of proteins expressed in the cells. As a second step labeled cells are lysed and lysates are incubated with a affinity beads for immuno purification of the proteins to be analyzed.
Next samples of labeled and purified proteins are run on sodium do desal sulfate, poly acrylamide gel electrophoresis and blotted to poly olaine fluoride membranes. In order to detect radioactive phosphate incorporation and protein levels, results are obtained that show leucine rich repeat kinases one and two, abbreviated L rrk one and L rrk two are phosphorylated in cells to comparable levels based on auto radiography and immuno detection. A main advantage of the metabolic labeling technique compared to other techniques such as phospho, immuno blotting, are that this technique allows the determination of total cellular phosphorylation levels of proteins including for proteins for which no phospho antibodies are available.
Second major advantage of this technique are that quantitative comparisons can be performed between different proteins in their total cellular phosphorylation levels. Demonstrating the procedure will be F gao, a technician from our laboratory To begin culture HEC 2 93 T cell lines according to standard culture conditions in dcos modified eagle, medium, or DMM with 8%fetal calf, serum and gentamycin. Expand plate and express triple flag tagged L rrk two protein via transfection or lentiviral vector mediated transduction as discussed in the text protocol.
When cells are 80 to 100%confluent, rinse them with prewarm phosphate free dmm. Under a laminar flow, prepare a falcon tube with 2.1 milliliters of phosphate free DM a m per six well plate of cells to be labeled with phosphorus. 32 orthophosphate.
Prepare the bench at which the experiments with ionizing radiation will be performed. The working space is covered by a spill mat upon which a protective liner of absorbent material is placed. Also provide for a perspex jar on the workspace and placed the tube of phosphate free medium inside.
Remove the lead lined container with the vial of phosphorus 32 labeled orthophosphate out of the refrigerator and bring it to the radioactivity bench. Monitor the container for external radioactive contamination using a Geiger counter dilute the phosphorus 32 labeled orthophosphate into the tube of phosphate free dmem. Add a concentration of 24 micro curies per milliliter.
Keep the tube in the perspex jar. Close the container with the remainder of the phosphorus 32 labeled orthophosphate before returning it to the refrigerator. Next, remove the six well plates with cells to be labeled from the incubator and place on the radioactivity bench.
Remove the medium supernatant and discard. Then add two milliliters of the phosphate free medium containing phosphorus. 32 labeled orthophosphate per well.
Place the culture plates into a perspex box and then monitor the container for external radioactive contamination using a geiger counter. Then transfer the perspex box with cells to a eukaryotic cell incubator dedicated to isotopic metabolic labeling and incubate the cells for one to 20 hours. At this point in the protocol, cells can be treated with compound if desired as instructed in the text protocol Following cell treatment and incubation, remove the medium from the cells and discard in into a waste collection tube which is placed in the perspex jar.
Rinse the cells two times with two milliliters of ice cold tris buffered saline. Discarding the rinse solution into the waste collection tube. Then add 0.5 milliliters of ice cold immunoprecipitation lysis buffer to each well and collect lysate by pipetting up and down.
In order to loosen all lysed cells, transfer the lysate to a micro centrifuge tube and incubate on ice at least 10 minutes before centrifusion as directed in the text protocol to isolate the protein of interest by immuno purification. First, transfer the micro centrifuge tubes with centrifuge lysates back to ice. Then pipette the supernatant into micro centrifuge tubes containing 10 microliters bed Volume of flag M two aro speeds.
Equilibrated as discussed in the text protocol, transfer the micro centrifuge tubes to a 50 milliliter tube. After transferring the micro centrifuge tubes to labeled 50 milliliter tubes on ice, place the samples onto a rotating device behind a perspex shield in the designated area of a cold room for end over end mixing at four degrees Celsius for one to 20 hours following incubation, spin down the protein bound flag M two aro beads in micro centrifuge tubes after discarding the supernatant into a waste collection tube. Proceed to wash the beads as discussed in the text protocol.
Then resuspend the washed beads in 40 microliters of IP sample SDS loading buffer Heat the samples in loading buffer to 95 degrees Celsius for two minutes before centrifuging for one minute at greater than 1000 times g to pellet the beads prepare the protein gel electrophoresis module on the radioactivity bench behind a perspec screen. Load the samples onto three to 8%T tris acetate SDS page gels including a molecular weight marker which is suitable to discern sizes of high molecular weight proteins before performing electrophoresis. After electrophoresis, remove the gel from its plastic casing and transfer to a container with western blotting transfer buffer.
Following preparation of the gel and PVDF membrane. Prepare the blotting sandwich on the surface of the semi-dry blotting module as described in the text protocol. Transfer the proteins at 15 volts for one to two hours after transfer.
Remove the PVDF membrane with blotted proteins from the blotting module and dry the membrane to perform auto radiography. First, expose the membrane to a phosphorescence plate for one to five days. Read the phosphorus 32 off of the exposed phosphorescence plate using a phosphorescence scanner and save the image as a high resolution tiff to detect protein levels via immuno detection.
Rehydrate the membranes by dipping them briefly into methanol and then transferring them to a shallow blott incubation vessel with PBS. Block the membranes in PBST containing 5%milk. Incubate the blots with anti LR K two antibody or Anti-Flag antibody and process further with appropriate wash steps and secondary antibody incubation.
Following antibody application, perform chemiluminescence detection to confirm the relative protein levels of LRRK two. Finally, quantifying corporation of phosphorus 32 in LRK two using diametric analysis as described in the text protocol Shown here are representative results for metabolic labeling of L Rrk one and L Rrk two in HEC 2 93 T cells. Depicted here is a representative auto radiogram of the phosphorus 32 incorporation as well as a representative western blot of L Rrk one and L Rrk two detection via their triple flag tags.
Radioactive phosphate incorporation is observed for both L rrk one and L rrk two. Upon quantification of the phosphorus 32 levels normalized to the protein levels as measured by density metric analysis of the immuno detection with Anti-Flag antibody. It was found that L rrk one had an average phosphorylation level which is lower than L rrk two under the conditions tested, although statistical significance is not reached, Don't forget that the working with radioactivity can be extremely hazardous and the precautions such as very personal protective equipment and working via procedures provided by the safety department of your institution should always be taken via performing this protocol.
