Today was a pretty typical day in lab. I completed a phosphorylation assay using 10% DMSO to make sure that the assay works with DMSO.
I started out the day by stopping the dephosphorylation reaction that ran overnight. The reaction mixture included dephosphorylated HipA, MnCl2, lambda-phosphatase, and buffer solution. I began by isolating the dephosphorylated HipA using a Ni-affinity chromatography column. After the protein was isolated, I needed to concentrate it up because a specific concentration is required for the phosphorylation assay. I centrifuged 3 mLs of the elution fraction at a time, concentrating the protein. Next, I needed to buffer exchange since the buffer used for the dephosphorylation reaction is different from the buffer required for the phosphorylation assay, so I centrifuged the protein three more times, diluting it by a factor of five with the new buffer each time, resulting in a 125-fold dilution. I checked the concentration of the protein and found it to be 0.71 mg/mL, indicating approximately 70% yield, which is pretty good. I left my protein in the fridge and headed to lunch.
After lunch, I began the phosphorylation assay. I have already done this assay twice, but today I needed to try it with 10% DMSO to make sure that it gives the same results as without DMSO. The compounds that I will test later are kept in 10% DMSO, so it is important to make sure that this will not affect my results and that the usual assays still work with DMSO. I diluted my protein to a concentration of 0.05 mg/mL in assay buffer and put 500 uL of this dilution into one tube, and 450 uL of this dilution into a second tube. I also added 50 uL of 100% DMSO to the second tube to create a concentration of 10% DMSO to mimic what the compounds to be tested later are stored in. I removed 20 uL samples from each tube to serve as my controls. Then I added 5 uL of ATP to each tube and incubated them at 37°C, beginning phosphorylation. I removed 20 uL samples from each tube after five minutes, fifteen minutes, thirty minutes, forty-five minutes, an hour, and two hours. After removing the samples, I immediately heated them at 99°C for five minutes to denature the protein and stop the reaction. I then added loading dye and stored them in the fridge for later when I will run the gel that will show me my results. The purpose of removing samples at different times is to demonstrate how HipA auto-phosphorylates in the presence of ATP over time. I will compare the results of the protein with and without 10% DMSO to determine if the DMSO affected HipA’s auto-phosphorylation or if it affects the phosphostain procedure that I will use to visualize the gel. If the two gels look the same, I am safe to use this procedure with the test compounds. If the gels do not look the same and the DMSO does in fact affect the assay, I will have some troubleshooting to do before I can test out the compounds.
Overall, a usual, but rewarding, day in lab. I can’t wait to stain the gels and see what my results are.