Western Blot

Consensus Approved Protocol 3/31/00- Contact Person is Kate Harrell

1. Soak the gel in 1X western transfer buffer @ room
temperature for 15 min.
2. Cut out a piece of immobilon filter. Wet in 100% MeOH. Then wash quickly one time for 2 minutes with about 200ml of 1X transfer buffer. Then soak in 1X transfer buffer for at least 10 minutes.
3. Cut out 6 pieces of 3mm paper each the size of the gel. (6 17cm X 19cm can be cut from one big sheet).

1. Check that all parts of the apparatus are clean. If not clean them.
2. Wash the SCOTCH-BRITE pads with water, soak in them in transfer buffer, squeeze away all dirty materials coming out of the pads, and soak in another volume of transfer buffer.
3. Place the tray on a level surface.
4. Place the bubble screens (2) on the bottom of the tray.
5. Place one of the electrodes on the bubble screens.
6. Place another bubble screen on the electrode with ribbed side down.
7. AT THIS POINT, THE TRAY SHOULD BE FILLED HALF WAY WITH TRANSFER BUFFER.
8. Place 2 SCOTCH-BRITE pads on the top of the bubble screen, AND SQUEEZE ALL OF THE BUBBLES OUT OF THE PADS!
9.  Wet the 3MM paper in the transfer buffer and carefully place the wetted 3MM paper on the top of the scotch pad making sure no bubbles have been trapped.
10. Repeat step 9 for two more pieces of 3MM paper. NO BUBBLES!
11. NOW YOU ARE READY FOR THE GEL.  IT IS GOOD TO CHANGE GLOVES AT THIS POINT.
12. Carefully place the gel on the 3MM paper, and remove the bubbles with great care.
13. Place the immobilon sheet SLOWLY from MIDDLE TO SIDES on top of the gel, again, no bubbles allowed.
14. Place a wetted 3MM paper on the immobilon, without trapping any bubbles.
15. Place two more wetted 3MM papers on the previous 3MM paper as in step.
16. Place 2 soaked Scotch-Brite pads on top of the 3MM paper.
17. Place a bubble screen with ribbed side up, and fill the tray all the way up with buffer.
18. Place the other electrode on the screen.
19. Place the plastic cover on the electrode.
20. Keep the tray level and slide the tray in to the tray holder.
21. AT THIS POINT THE GEL SANDWICH SHOULD FIT TIGHTLY INTO THE APPARATUS. IF IT DOES NOT YOU SHOULD USE THREE SCOTCH BRITE PADS ON EACH SIDE RATHER THAN TWO.
22. Tip the whole assembly to its vertical position, run @ 15 V/ 370mA for 30 min at 4C. The positive electrode should be on the immobilon side of the gel/immobilin pack.

1. Disassemble the western transfer assembly.
   
2. Block with 5% w/v powdered milk in PBS for at least 1hr @ room temperature. If kept longer than 3hrs, place at 4C.
   
3. Wash the filter quickly in PBS (no milk) to get rid of loose milk particles etc.
   
4. Add 1st antibody. The incubation conditions will vary as will the amount of solution used, depending on the particular antibody and the vessel used for blotting. Typical protocols are 1 hr at RT or overnight at 4C with 20-50 ml of solution. Seek guidance from someone who has used the antibody before as to how what dilution to use. Solution composition: PBS w/ 2% milk and 0.05% tween 20 + antibody.
   
5. Remove the 1st Ab and save it in -20 degrees C freezer.
   
6. Wash the blot quickly 1X with PBS w/ 2% milk and 0.05% tween 20. Then 3X more for 20 min each. (Use 10X the volume used in 4) for each wash).
   
7. Add 2nd Ab in PBS/2% milk/.05% tween 20. Again ask about concentration and appropriate method of development. The development method will determine what antibody to use. Typical dilutions are between 1/1000- 1/10,000. Usually 1hr at RT is sufficient. (SEE PROCEDURE BELOW FOR USING RADIOACTIVE PROTEIN A instead on 2nd Ab).
   
8. Wash the blot with PBS/0.05% tween 20 for 3X@ 20 min each.
   

For alkaline phosphatase:

1. Develop in: 40 ml l X alkaline phosphate buffer/264ul NBT/132ul BCIP for about 10 to 15 min at RT.
   
2. Wash the blot with ddH20.

For ECL (Amersham RPN2106):

1. Place about 6 ml each of reagent A and B in a dish and soak the blot for 1 minute with rocking.
   
2. Wrap blot in plastic wrap face down and tape to exposure cassette. Take cassette to darkroom along with film and expose for about 1 minute. Make determination of the appropriate exposure based on this exposure. (Range 15 seconds to 15 minutes). Mark the position of the molecular weight markers on the film by lining up the developed film with the blot.

THIS IS RADIOACTIVE SO TAKE GREAT CARE AND FOLLOW THE PROTOCOL EXACTLY

1. Take container with the I-125 Protein A to fume hood and it open there. The I-125 is conjugated to Protein A.  Protein A binds to the Fc portion of the primary antibody, so in this way it acts like a secondary antibody. Although there is very virtually no free I-125 open the stock bottle only in the hood. (FEDERAL REGULATIONS REQUIRE THAT THE STOCK BOTTLE BE OPENED ONLY IN THE HOOD)
2. Under the hood remove the desired amount from container and put in a tube with PBS/0.05% tween (PBST) (usually ~10ul (~10uCi) per 10ml PBST) and put the lead stock container back in fridge
3. Add to blot for ~1 hour (this can be done on a bench top rocker but be sure to label container as radioactive)—> the radioactive liquid can be saved for use next time at -20C. MARK THE TUBE WITH RADIOACTIVE TAPE!
4. Wash as normal after 2ndAb ****But be sure to discard wash waste in the radioactive I-125 liquid waste container. It is easiest to collect wash waste in a beaker under the hood until all washes are done then empty waste all at once.
5. After washes place blot in baggy or wrap and place in phosphoimager cassette. A few hours exposure is usually enough.

Important

• Make sure to sign out the I-125 in the Radiation Safety book and record waste disposal.
• Do a survey after usage to make sure you did not spill any I-125 and add this to the Radiation Safety book under I-125 surveys.

Western Transfer Buffer (WTB)
30.3 g Tris Base
144g Glycine
2 Liters Methanol
Make up to 10 Liters total with water

Alkaline Phosphatase Buffer (APB)
.1M Tris-HCl, pH 9.5, .1M NaCl

Nitro Blue Tetrazolium (NBI)(N-6876)
50mg/ml in 70% dimethylformamide
5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)(Sigma B-6149)is made up at 25 mg/ml in water.