Electrophoresis and Western blot protocols Removing Heads: 1.

Remove samples from the freezer and keep on dry ice along with a metal surfac e. Vortex the sample several times to remove the heads. 2. Place the now chilled metal surface underneath the microscope. Use the "cheap " forceps to remove any heads that remain attached. Place all of the heads into an eppendorf tube. 3. Prepare lysis buffer (___mL Invitrogen Tissue Extraction Reagent #1 with ___L phosphatase inhibitor). Add 100 L to the W1118 and TOP3 heads, and 60 L to all othe rs. 4. Grind down to break up tissue. 5. Repeat for all samples. 6. Spin down for ten minutes at 15,000 rpm and move the supernatants to another set of tubes. -Measure the concentrations of the samples using the Coomassie assay: 1. Turn on spectrophotometer. Allow to warm up. 2. Add 2mL of Coomassie reagent to the spectrophotometer tubes. Add 5L of sample to each one. Make sure to include a blank. 3. Select Protein Assay -> Bradford -> Formula -> Enter 4. Test blank first. 5. Test all other samples in order. Make sure to record the concentrations on pa per. 6. Setting the smallest concentration to 10L, calculate the amounts of protein ne eded to load all the wells evenly. 7. Calculate the amount of buffer needed to bring the total volume of each sampl e to 42L. -Running the gel: 1. Prepare the loading buffer: 475L Laemmli Sample Buffer + 25L beta-mercaptoethan ol. 2. Add to the samples using the calculations from the step before. 3. Heat samples for five minutes, making sure the caps are closed and covered wi th a heavy metal piece. 4. Spin at 15,000 rpm for one minute. 5. Break open the packages for the gels. Remove combs and the white strip at the bottom. Wash with DI water. Clamp into place evenly. 6. Pour the running buffer (Tris/Glycine/SDS buffer) to cover both ends of the g el. 7. Load the Benchmark Protein ladder (blue color) on the left side and the Novex Sharp Prestained Ladder (purple color) on the right side by mixing 10L of the la dder with L of the sample buffer. Fill in a blank between the ladders and the sam ples with 20L of sample buffer. 8. Add 19L of each sample into the appropriate wells. 9. Attach the tops of the box, connect the wires, and set to run at 120 V for 95 minutes. 10. Try not to take a nap. -Transferring: 1. Take two pieces of memory, and wet with DI. Then cover with transfer buffer. Set aside. 2. Lay the mesh in the buffer and roll to remove bubbles. Add two pieces of filt er paper to the same container. 3. Dump the running buffer and wash the gel with DI. Use a scalpel to cut along the bottom. Break the gel container by using the spatula like a lever on the edg es. Carefully remove the gel and rest it in the buffer, on top of one of the pie ces of filter paper. Make sure the colored ladder is on the LEFT side so that it will transfer with the correct orientation. 4. Add the sheet of memory on top. Then add the second piece of filter paper, th

e second mesh, and finally lock the entire thing in place with the transfer box. Make sure that the black side of the box is on the bottom, and that the black s ide ends up facing the black wall of the outside box. 5. Run at 17 V overnight. -Blotting: 1. Mix 2.5 g dry milk with dilution buffer to form 50 mL of 5% solution. 2. Cover the memories with the red stain for 1 min. Return the stain to the tube for future resuses. Wash three times with DI. Label major benchmarks on the pro tein ladder as well as their molecular weights (which ones?) 3. Cut into two pieces? Set the pieces in milk for 1 hr. 4. Dilute antibodies appropriately. (Which antibodies?) 5. Put pieces in sealed bags with their antibodies, removing bubbles. Rock for 2 hours. 6. Remove antibodies and keep in the used antibody box in the 4 degree refrigera tor. 7. Wash three times with washing buffer, 8 minutes each. 8. Add secondary antibodies, incubating with 3mL of the diluted antibody solutio n for 1 hr in sealed bags, rocking. 9. Wash the same way as before.***END OF FILE***