Running a Protein Gel

Materials:

1. GLOVES
2. One clear gel bottom
3. One gray gel center with power connections on top
4. One gel top with wire connectors
5. four red clamps
6. gel(s) to be used (if only one gel is being run grab another plate for the opposite side to resist spread of the buffer

Procedure:

1. WEAR GLOVES WHENEVER USING ACRYLAMIDE
2. Place the gel bottom on the bench. Place the gray gel center piece with the power connections on top in the center of the gel. Place your gel on one side with the glass plate facing you. Use two red clamps to hold the gel in place with the longer half of the clamp touching your gel.
3. If you are doing two gels at once place your other gel on the other side in the same manner. Otherwise take the other extra plate and clamp it onto the gray center piece.
4. Pour the cathode buffer down into the small space between your gel and the gray center piece being careful not to spill buffer into the lower section of the gel bottom.
5. Pour the anode buffer into the gel bottom putting enough in just to cover the little humps.
6. Take a marker and mark where the wells are on the glass plate to make it easy when loading samples.
7. Remove the comb from the gel
8. Load all the samples into the gel. Remember to put a Molecular Weight Standard (in the third section in the -80 freezer) in the gel and put it in the far left well.
9. If doing a gel to test for purification be sure to load a sample of the supernatant (the stuff you loaded into the loop with the syringe), a sample of the flow through, and all the samples from the test tubes that you chose.
10. Once all of your samples have been loaded carefully place the top with the wire connectors onto the apparatus being sure to match the colors for positive and negative.
11. Turn the timer knob to 1 hour
12. Set the current at about 65
13. Make sure the voltage knob is turned all the way on.
14. If the voltage reads a very high number such as 500 there is something wrong with the apparatus and it might be that you did not add buffer.
15. Keep an eye on the gel and once it has run a decent distance you can turn the timer knob to the off position.
16. Unplug the wires from the voltage machine and take the entire apparatus over to the gel sink.
17. Turn the warm water on so that you can take apart the apparatus and rinse it as you go.
18. Take the top of the gel apparatus off, rinse and put in the drying rack
19. Undo the clamps for the gel that you are saving and carefully remove the gel.
20. Carefully slide one of the gray separators out of the section between the glass plate and the siliconized plate.
21. Take the gray separator and carefully stick it in between the glass and the gel. Then slowly turn the separator so that the glass begins to pop off.
22. Then using the separator start to separate the gel from the siliconized plate. Once you have separated one of the corners use your fingers to grab the corner and peel the gel off of the plate.
23. Place the gel into one of the large Petri dishes that is near the gel sink and cover it with one of the covers.
24. Finish taking apart the apparatus and rinse every part placing them in the drying rack to dry.
25. Then take your Petri dish with your gel over to the blue comassie stain.
26. Pour enough Comassie stain into the Petri dish so that your gel is completely covered and be sure not to fill more than 1/3 of the Petri dish so that it doesn’t slosh around on the shaker.
27. Place the whole Petri dish on the shaker and let it stain for about 20 – 25 minutes.
28. After the staining is complete holding the gel with one of your fingers pour the excess stain back into the bottle containing the comassie stain.
29. Take two Kimwipes, crumple them into a ball, and place them into the Petri dish with your gel.
30. Pour enough destain (in a dispenser over by the gel sink) to cover your entire gel. Again do not fill more than 1/3 of the Petri dish to prevent spillage on the shaker.

31. Cover the Petri dish and place back on the shaker. The gel will be ready for analysis after about 10 minutes.