Cellular Transfection and Immunoprecipitation
Before proceeding with the experiments outlined below, all kinase pocket mutants should be characterized for their expression, localization, specific activity, and substrate specificity. The results should be compared with those obtained from the wild-type kinase to ensure that the mutation does not significantly alter these characteristics.Grow COS-1 cells in DMEM supplemented with 10% FBS. Plate cells the day before transfection at 4 x 105 cells per dish in 60-mm tissue culture dishes. Incubate at 37°C and 5% CO2 overnight.
Transfect cells with 2 µg of expression plasmid using Lipofectamine, according to the manufacturer's specifications.
We have found that a 1:4 ratio of DNA to Lipofectamine is optimal for transfection of COS-1 cells.
Incubate the cells for 24-72 hours at 37°C and 5% CO2. The rest of step 3 and step 4 are optional and can be varied depending on the kinase of interest. For mitogen-activated kinases, serum-starve transfected cells by washing twice with room-temperature phosphate-buffered saline (PBS) and placing in serum-free media for 4-12 hr prior to harvest.
Stimulate the cells for 10 min before harvest with an agonist that activates the kinase of interest. Harvest the cells at 24-72 hr posttransfection.
Wash the cells twice in cold PBS on ice. Drain the dishes well. Add 0.5 ml of M2 lysis buffer and scrape each dish of cells into microcentrifuge tubes. Vortex. Let the cells lyse on ice for 15 min, vortexing occasionally. Centrifuge at 15,000g and 4°C in a microcentrifuge for 15 min. Transfer the supernatant to a fresh tube.
Perform a protein assay on the cell lysates using a BCA assay kit from Pierce. We typically obtain 0.5 mg of protein per 6-cm dish.
Prepare the antibody for the immunoprecipitation (Harlow and Lane 1999). We use M2 agarose for FLAG-tagged ERKs. If the antibody to the epitope tag on the protein is not preconjugated, prebind the primary antibody for your epitope tag to protein A- or protein G-conjugated agarose or Sepharose CL-6 beads. If necessary, use a secondary antibody to link the primary antibody to the beads (Harlow and Lane 1999). The amount of antibody to use will be antibody- and protein-specific. For the small-scale immunoprecipitations described in step 8, 0.1 µg of antibody per immunoprecipitation should be sufficient. Batch prebinding can be performed with enough antibody and conjugated beads for all of your reactions. Prebinding should be performed in 1 ml of M2 cell lysis buffer for 1 hr at 4°C with constant agitation. Additional unconjugated Sepharose CL-6 beads can be included in the prebinding to increase the bead volume for each kinase reaction. We find that a total of 30 µl of bead volume is optimal for each reaction. After prebinding, centrifuge the antibody mixture at 15,000g in a microfuge for 30 sec. Aspirate the supernatant and wash the beads twice in M2 lysis buffer to remove unbound antibody. Aspirate the wash buffer. Resuspend the beads in an equal volume of M2 lysis buffer to make a 1:1 bead:buffer slurry.
Aliquot 50 µg of cellular protein (as determined in step 6) into fresh microcentrifuge tubes, using one tube for each reaction (use more or less protein, depending on the abundance and activity of your kinase). We typically perform kinase reactions in duplicate to control for variability in the assay. Add M2 lysis buffer to a total volume of 750 µl.
Aliquot the preconjugated antibody (60 µl of 1:1 bead:buffer slurry from step 7) into each of the tubes containing cell lysate. Incubate at 4°C for 2 hr with gentle rocking.
Centrifuge the samples at 15,000g at 4°C for 30 sec. Aspirate the supernatant. Wash the beads three times with 1 ml of cold M2 lysis buffer, followed by two washes with 1x kinase buffer. Aspirate the final wash buffer down to the beads. Keep the samples on ice.
Just before starting the kinase reaction, aspirate the residual kinase buffer with a 27 gauge needle attached to a 1-cc syringe and a vacuum flask. Keep the beveled side of the needle facing the wall of the tube to prevent aspirating any beads. Use this immunoprecipitated protein for the analog inhibition assay and the substrate phosphorylation assay.
Analog inhibition assay and substrate phosphorylation assay: 2x 50- µg aliquots of cellular protein are required for the analog inhibition assay and a further 2x 50 µg are required for substrate phosphorylation.
Add 40 µl of kinase reaction mixture to each tube on ice. (Note: Kinase reaction mixture A is for analog inhibition assays. Kinase reaction mixture B is for the substrate phosphorylation assay.) Mix gently by flicking the tube with your finger.
Incubate samples in a 30°C water bath for 10 min. Place the samples on ice and immediately add 40 µl of 2x Laemmli sample buffer to stop the reaction. Vortex the samples.
Heat the samples for 4 min in a boiling water bath or heating block. Centrifuge the samples at 15,000g at room temperature for 30 sec.
Load the supernatant on an SDS-polyacrylamide gel and electrophorese.
Transfer the gel to nitrocellulose membrane.
Place the membrane in a glass dish. Stain the membrane for 5 min with Ponceau S solution. Remove the stain and destain the membrane twice, for 5 min each, with 1% acetic acid.
If [y-32P]ATP or [y-32P]ATP analog is used, cut the substrate bands out of the gel and count them individually by dry Cerenkov counting on a scintillation counter. If a phospho-specific antibody to the substrate is available, leave the blot intact and immunoblot with the antibody (Harlow and Lane 1999).
For the analog inhibition assay, calculate the percentage of labeled phosphorylation from each reaction. The cpm from duplicate assays should be averaged, and the value obtained from the kinase reactions that did not contain ATP analog should be set to 100%. Then compare the average counts from the other kinase reactions to those of the control reaction and express the ratio as a percentage.
For the substrate phosphorylation assay, use the mutant kinase/ATP analog pair that gives the highest phosphorylation, either through immunoblotting or radioactive measurements, for future studies.
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