The overall goal of this assay is to measure the enzymatic activity of protein kinases. This method can help answer key questions in the kinase signaling field, such as how active a kinase or kinase mutant is, its specificity, and whether its activity is sensitive to different cell treatments. The main advantage of this technique is that it is both versatile and quantitative.
Demonstrating the procedure will be Steve Stippec, a research scientist at the Cobb Laboratory who has a well of experience with this assay. To begin, add two microliters of one milligrams per milliliter antibody to 200 microliters of cell lysates and incubate at four degrees Celsius for one hour while rocking. Wash protein A-Sepharose beads two to three times by adding lysis buffer and then touch spinning at four degrees Celsius for 30 seconds to one minute at 5000 times g, then remove the supernatant with a pipet and re-suspend the beads in buffer.
Next, add 30 microliters of 50%slurry of protein A-Sepharose beads and lysis buffer to the lysates. Incubate the lysates at four degrees Celsius for one hour while rocking. Touch spin at four degrees Celsius to pellet the beads and remove the supernatant.
Wash the beads three times with one milliliter of bead wash buffer, followed by touch spinning, then wash the beads once with one X kinase reaction buffer. After touch spinning, remove as much buffer as possible without removing the beads. To initialize the assay, add the entire reaction mixture to the kinase sample.
Incubate the reaction at 30 degrees Celsius for five minutes to one hour depending on the activity of kinase being assayed. Stop the reaction by placing it on ice and adding 7.5 microliters of five X Laemmli sample buffer, then heat the reaction at 100 degrees Celsius for three to five minutes in the heat block. After touch spinning the sample, load 20 microliters per well on a 10 to 15%SDS-PAGE gel.
Make sure to keep the gel apparatus shielded to limit exposure to phosphorus-32 because it is a high energy beta emitter. Run the gel long enough to separate kinase and substrate. Following the run, remove the gel from glass and aluminum plates, then place the gel in 50 milliliters of Coomassie stain for one hour on an orbital shaker set to 50 RPM.
For this step, use a container that is slightly larger than the gel itself. To destain the gel, move it from the stain to the fixing solution. Place pieces of foam or knotted laboratory wipes in the container with the gel to absorb the Coomassie dye.
Rock the gel in 600 to 700 milliliters of fixing solution overnight on orbital shaker at 50 RPM. The next day, remove the gel from fixing solution and soak it in 200 milliliters of methanol for one to two minutes with gentle agitation until the gel turns milky white. This will help prevent cracking during the drying step.
Next, wet a roughly 14 centimeter by 14 centimeter piece of qualitative filter paper with methanol and place it onto a slab gel vacuum dryer. Lay the gel down onto the filter paper, front side facing up. Carefully cover the gel with plastic wrap, then turn on and run dryer and apply vacuum.
Once the vacuum has been attained after only a few seconds, roll out air bubbles with a soft rubber brayer. Continue the run for 90 minutes. Remove the dried gel and attach an autorad marker, such as a phosphorescent ruler or dot to the filter paper on the side of the gel and place it in a cassette containing an intensifying screen.
Use a Geiger counter to check the intensity of the signal. For weaker signals, exposure at minus 70 degrees Celsius to minus 80 degrees Celsius with an intensifying screen will increase the film band density. In a darkroom, place a piece of film on top of the dried gel inside the film cassette containing an intensifying screen.
If the CPM count is 100 or below, try an overnight exposure at minus 70 to 80 degrees Celsius. Alternatively, if the count is closer to 10, 000, start with a one hour exposure and optimize from there. At the conclusion of the exposure, remove the film before developing it in a medical or X-ray film processor.
Mark the bands corresponding to the protein standards on the film. Also, label the protein standards and the reaction lanes for future reference. Excise the bands from the gel that correspond to the bands of interest on the film.
Place the bands in seven milliliter scintillation vials and add four milliliters of scintillation fluid. Count the bands with a liquid scintillation counter. Confirm that the counter is set to monitor the correct energy spectrum window for phosphorus-32.
Proceed to analyze the results as described in the text protocol. Purified ERK2 fractions were used in a kinase assay with MBP in the presence or absence of MEK1R4F, a constitutively active stimulator of ERK2 kinase activity. In a similar experiment, an MBP kinase assay was used to measure activity of recombinant ERK2 mutants purified from bacterial cultures relative to wild type protein.
Although ERK2 was able to phosphorylate MBP when stimulated by MEK1R4F, ERK2 kinase activity is dramatically abrogated by mutation of either the catalytic lysine or a threonine proximal to the canonical sites of dual phosphorylation. ERK2 T188D and ERK2 T188E display marginal kinase activity toward a small flexible peptide. However, they are unable to robustly phosphorylate the known ERK2 substrates, Nup153 and PDX1, as seen with wild type ERK2.
Once mastered, this technique can be done in one to two days if it is performed properly. After watching this video, you should have a good understanding of how to use radiolabeled ATP to assay the activity of protein kinases to better understand the signaling networks in which they reside. While attempting this procedure, it's important to remember to optimize the amino precipitation protocol for pulling down kinases of interest to account for equal protein amounts and to measure concentrations of recombinant protein to ensure accurate comparisons of kinase activity.
After its development, this technique paved the way for researchers in the field of cell signaling to explore specific transduction pathways and their roles in homeostatic regulation as well as their contributions to disease progression. Don't forget that working with radioactive materials can be extremely hazardous, and precautions, such as wearing personal protective equipment should always be taken while performing this procedure.
