Note: Sessions 7 and 8 can be combined into one laboratory day if you would prefer one long session instead of two short sessions.
During Sessions 7 and 8 you will analyze the purified H396P Abl(229-511) by SDSPAGE gel electrophoresis. You will also determine the concentration of your protein domain after purification and dialysis.
1.) Running an SDS-PAGE gel Remove your cast gel from the storage container, remove the white strip of tape from the bottom of your gel, and place the cassette in the gel running apparatus. Two gels fit in a gel box, so two groups of students should use each box. Fill the apparatus with 1X electrophoresis (TANK) buffer such that the section between the two gels is filled to the top and the outer sections are filled halfway with buffer. Carefully remove the gel comb. Using gel-loading pipette tips on a 20p pipette, load 5 uL of protein ladder (provided by your TA) into a corner well of your gel. The protein ladder consists of proteins with known molecular weights conjugated to visible dyes and does not need to mixed with the sample loading buffer. See the posting on the -20 freezer or the figure below to identify the molecular weight markers in the protein ladder. In the next two wells, load 10 uL of the pre and post-induction samples. In the final 7 wells, load your Ni-NTA elution samples. Run the gel at 200 V until the blue dye reaches the bottom of the gel (approximately 45 to 60 min).
Once the gel running is complete, disassemble the gel apparatus, and carefully pry the cassette apart with a spatula. Transfer the gel into a container for staining. Running a light stream of water over the gel can help prevent gel tearing during cassette separation and gel transfer. Add enough Coomasie blue staining solution to cover the gel (10 to 25 mL), and place the container on the gel rocker. Rock the gel in the stain for 30 minutes. Pour the used staining solution into a designated bottle, and rinse the gel with water. To destain, cover the gel with fast destaining solution and add a balled-up Kimwipe to the corner of the gel container. Rock the gel at room temperature for 30 to 50 min, then replace the fast destain with the slow destain solution and rock the gel overnight. For the overnight destain, add a fresh Kimwipe and cover the container with saranwrap to avoid excessive evaporation (especially during the winter months).
The H396P Abl kinase domain should appear as a band at approximately 33 kDa. Depending on the protein concentration, some impurities may be visible in the more concentrated elution fractions. In the literature, these impurities have been reduced or eliminated by running a second column. However, we will use the singly purified protein for our assays, since we are interested in comparing the activity with vs. without inhibitor rather than determining absolute activity.
(lab open 1-2 pm) After destaining, pour the destain solution in a designated waste bottle and check your gel for protein bands. You should rinse the gel with water and take a digital picture for your laboratory report. Once you have a decent picture of your gel, you may discard it.