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Western Blot Protocol: Sample Preparation1. Aliquot samples to a standardized amount (i.e.

25 g) that does not constitute an excessive volume (usually you want to aim for around 30 L to comfortably load the wells). 2. Make sure to account for the volume of 4x loading buffer that needs to be added to the samples so that they run properly (This loading buffer should account for the volume of your total sample to be loaded i.e. 7 L of a 28 L sample). 3. The samples can be frozen in loading buffer immediately after doing a protein assay so that they are ready to run at a future date. 4. When you are ready to run your Western Blot gels, heat the samples for 5 minutes at 80100 C (there is a hot block in the Cremo Lab). 5. After heating, briefly spin the samples down in the table top centrifuge for about 10 seconds. 6. The samples are now ready to be loaded on the gel. Running the Gel1. Obtain the appropriate gels from the deli-style refrigerator in the adjacent room (The gels come in a large variety with different percentages and numbers of wells keep in mind the sizes of the proteins you want to look at and how many samples you have at what volume when deciding what gel type to use). 2. Remove the gel comb and the white tape sealing the foot of the gel. 3. Wash the wells briefly under dH2O to remove any junk that has accumulated in them. 4. Secure the gels in the gel apparatus. 5. Make 500mL of Running Buffer (the stock is 20x) per apparatus being used (2 gels fit in 1 gel box), and completely fill the center chamber between the gels. The outside part of the running apparatus only needs enough buffer to cover the slot where the foot of the gel is. 6. Write out and plan how you would like to load your gel ahead of time (making sure to include a Molecular Weight Marker) and follow this diagram when loading your gel. 7. Run the gel at 90V for 90-120 minutes, or until the dye from from the loading buffer is at or near the foot of the gel. 8. Rinse the apparatus well with dH2O, carefully hanging the center piece with the electrodes to dry so the wire is not being harmed and remove the gels (keep track of which is which if you are running more than one). 9. The gels are now ready for the transfer steps which should immediately follow (I would not recommend storing the gels after running for more than 1 hour). Transferring the Gel to Membrane1. You can use Nitrocellulose or PVDF. Nitrocellulose gives a lower background signal, but can only be stripped and reprobed 1 or 2 times before becoming unusable. PVDF binds protein very well and can be stripped many times, but it does have a higher background signal than Nitrocellulose. Also, if you are using PVDF, you must wet it in pure methanol before placing it in the regular transfer buffer. 2. While your gel is still running, make 1L of Transfer Buffer for each Transfer Module you intend to use (you can fit up to 4 gels into 1 Module). The Transfer Buffer Stock is 20x, but requires 20% methanol (i.e. 50mL of the 20x stock, 200mL methanol and 750mL dH2O for 1L). 3. Soak your membranes, filter paper and sponges in transfer buffer and make sure they are all thoroughly wetted (bubbles and dry patches will interfere with the transfer and you could end up ruining a publication-quality membrane with one tiny area of bad transfer). 4. Remove the gel from between the gel plates and allow it to equilibrate in transfer buffer for 2 minutes.

5. Create a transfer sandwich by following a pattern of sponge, filter paper, gel, membrane, filter paper, sponge (again, up to 4 gels can be placed like this in one module). 6. Place the completed sandwich in the Transfer Module, making certain that the appropriate membrane is closer to the positive electrode than the gel (the positive electrode comprises the flatter piece of the module). 7. Transfer at 35V for 1 hour if the gel was 1.0mm, or 90 minutes if it was 1.5mm. 8. Once the transfer is complete, wash the apparatus with dH2O and carefully hang it up to dry, disassemble the module washing it and the sponges and hanging them to dry and collect your membranes (the gel and filter paper can be discarded, unless you want to Ponceau S Stain your gel to make sure all the protein transferred). 9. Make sure your gel appears to have transferred to the membrane well. If there is any doubt, stain the membrane and/or the gel with Ponceau S to see. If neither stains, you may have set up your transfer in the wrong orientation and the protein was lost. Western Blotting (General)1. While the gels are transferring to membrane, make up the appropriate blocking buffer and primary antibody dilutions. For most blots, 5% milk in TBS-T is used for blocking and 3% milk in TBS-T is used to dilute the primary antibody (some antibodies require very specialized protocols though). 10mL is usually a good volume for the antibody dilutions and wash volumes. 2. Block the membrane for 1 hour in 5% milk TBS-T at room temperature on the shaker. 3. Add the appropriate dilution of primary antibody in 3% milk in TBS-T overnight on the shaker in the cold room. 4. Wash the membranes 3x with TBS-T for 5 minutes at room temperature on the shaker. 5. Add secondary antibody at 1:5k in 3% milk in TBS-T and allow it to sit on the shaker for 2 hours at room temperature. 6. Wash the membranes 3x with TBS-T for 5 minutes at room temperature on the shaker. 7. Make the ECL mixture (it is a 1:1 mix of the 2 reagents). I usually make 10 mL and use 5 mL per blot if there are more than 2 blots, you can transfer the ECL from the previous blots into the others as it is good for about 20 minutes after being mixed. 8. Add the ECL mixture to the membranes and keep them covered with it by gently shaking in your hand for 1 minute. 9. Poor the ECL onto subsequent blots or down the drain if you are done. 10. Remove any excess fluid from the membranes and place them in saran wrap so that only a single layer is on the face of the membrane with the protein. 11. Take the wrapped membranes down to the dark room to expose to film for developing (the dark room is in the Mastik Lab).