The overall aim of this procedure is to assay the kinase activity of leucine rich repeat kinase two. This is accomplished by first preparing a kinase assay mixture with recombinant LA two and a myelin basic protein substrate. Next radio labeled a TP is added and the mixture is incubated for one hour.
Then the proteins are denatured, separated on a poly acrylamide gel and subjected to western blot. Finally, the membrane is exposed to a phospho screen and the activity is measured. Ultimately, results can be obtained that show that LA two is active and how mutations alter its activity through auto phosphorylation and phosphorylation of MBP.
Visual Demonstration of this method is critical as the radioactive steps are difficult to learn due to the safety precautions that need to be taken. Before beginning this procedure, refer to the written protocol regarding the proper use and disposal of radioactive isotopes to prevent the spread of radioactivity. Prepare all reaction mixtures in 1.5 milliliter sample tubes with screw caps containing an O-ring.
Begin the assay by first thawing wild type LA two protein as well as variants on ice. Then mix the following ingredients on ice. 10 nanomolar of kinase, 0.5 micrograms per milliliter, myelin basic protein 0.5 microliters of 10 times kinase buffer, and enough water to bring the volume up to 50 microliters.
After preparing the work area and or personnel for working with radioactivity, set heating blocks to 30 degrees Celsius and 100 degrees Celsius respectively. Remove 32 PATP from the minus 20 degrees Celsius freezer. Scan the outside of the container prior to use and th it behind a perspex screen.
Using the Geiger counter, assess the radioactivity of the 32 PATP to each reaction on ice. Add one microliter of 32 PATP and 10 micromolar of cold. A TP Use a per pet to mix well.
Then pulse centrifuge the reactions to bring the liquid to the bottom of the tube, thereby minimizing the risk of contamination. Next, remove a 15 microliter aliquot as a zero time point and terminate the reaction by adding five microliters of four times SDS sample buffer. Then denature the aliquot at 100 degrees Celsius for 10 minutes.
Incubate the remaining sample at 30 degrees Celsius for 60 minutes. Then remove a second 50 microliter aliquot. Add four times sample buffer and incubator 100 degrees Celsius for 10 minutes.
To resolve the reactions on SDS page, load 20 microliters of each sample onto a 10. Well, four to 12%Biss poly acrylamide gel along with a lane of protein standard. Run the gel with mops running buffer at 160 volts for 90 minutes or until the DIA front reaches the end of the gel.
Transfer the protein to PVDF membrane at 25 volts for 16 hours according to the steps outlined in the written protocol. Once the membrane has dried, place it between two cellulose acetate sheets and expose it to either a phosphorous screen or an x-ray film to detect radiolabeled protein exposure. Time can run from several hours to over a week depending on the specific activity of the radioisotope used and the enzyme kinetics of the reaction.
If using a phospho screen, save the image is a high resolution TIFF or bitmap file for film. Use a desktop scanner to first scan the film. Use image J for image analysis here.
Results for the assay carried out using wild type lark two as well as the G 2 0 1 9 S overactive kinase and D 1 99 4 A kinase dead variance with myelin basic protein as the phosphate scepter as shown auto phosphorylation of Lark two is visible at around 200 kilodaltons with multiple bands representing phosphorylated MBP visible from 20 to 40 kilodaltons. Note the absence of auto phosphorylation in the D 1 90 94 a lane and increased phosphorylation due to the G 2 0 1 9 s mutation. Residual phosphorylation of MVP in the kinase dead lane may be due to incomplete ablation of the kinase activity of Lark two by the D 1 99 4 A mutant or may reflect the presence of trace contaminating kinases in the reaction.
Don't forget that working with radiation can be extremely hazardous and protective measures, such as shielding should always be used when undertaking a procedure such as this.
