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Western Blot

Warning: Acrylamide is a neurotoxin. Wear gloves at all time while handling gels!

Pouring the Gel

  1. Pour resolving gel about 3/4 of the way up the plate. Be sure there will be space between the bottom of the comb and the top of the resolving gel. Carefully overlay the resolving gel with H2O and let gel polymerize in a vertical position at RT for 30 minutes.
  2. Drain the H2O off and pour the stacking gel on top of the resolving gel and insert comb. Add extra gel if necessary to eliminate any bubbles at the top. Let polymerize for about 30 minutes at RT.
  3. Remove comb and bottom spacer. Rinse with H2O from tap and set up gel apparatus. Pour 1X TGS in top and bottom reservoirs of gel apparatus. Use syringe to flush out wells of any unpolymerized acrylamide. Also use bent needle syringe along bottom of gel to be sure no bubbles are stuck along the bottom of the gel.

Loading Samples

  1. Heat samples (in Laemmli sample buffer) at 100°C for 3 minutes. Quick spin and load. Be sure to load marker as well.
  2. Run gel. Voltage and time will vary according to the size of the protein in question and the percentage of the gel. Ask for advice from experienced colleagues.
  3. When gel has completed its run, take down gel. Remove top glass plate carefully, and, using a paper towel, remove the stacker layer of the gel. Often, you can cut the top left corner of the gel for orientation.

Transferring the Gel 

(for semi-dry blotter)

  1. Pre-equilibrate gel in 10% methanol:1X TGS on a slow shaker while you set up the rest for transfer:

    Cut 12 pieces of Whatman® paper and 1 PVDF in the same size as the gel. Make sure to wear gloves while handling these.

    Wet PVDF in methanol for ~2 minutes, then put in H2O for 5 minutes on shaker to hydrate.

    Switch PVDF into transfer buffer (10% methanol:1X TGS)

  2. Take out the blotting plates and individually place 6 pieces of Whatman paper, pre-wet in transfer buffer. Be sure there are no bubbles.
  3. Lay gel on Whatman papers face down.

    You can use the last Whatman and float the gel on to it to transfer to the stack.

  4. Place PVDF on top of gel and use an ink pen to mark the sizes of the marker lanes onto the blot. You can also mark lanes if want to.
  5. Place the last 6 pre-wet Whatman papers on top of the PVDF. Then place top plate of blotter and screw down to finger-tight.
  6. Run transfer at 300 mA for 45 minutes to 1 hour. Again, this may vary depending on the size of proteins of interest and the percentage of the gel. In addition, the percentage of methanol in the transfer buffer can range anywhere from 5%-15%. This will affect how long you transfer.

Western Blot

  1. While the gel is transferring, make up 1% instant milk/TBST blocking solution.

    This can go up to 5% milk for blocking, depending on antibody.

  2. Transfer blot with tweezers and gloved hands into blocking solution and let block for 1 hour at RT, shaking. You can do this in a Tupperware® container or Seal-a-Meal® bag.
  3. Incubate with primary antibody (Ab) diluted in blocking serum O/N shaking at RT. (Some people do this at 4°C as well). If the blot is in a Seal-a-Meal bag, try to keep as few bubbles as possible and use a minimum volume to conserve Ab.
  4. Wash blot 5X for 5 minutes in TBST at RT, shaking.
  5. Incubate in secondary Ab conjugated to horseradish peroxidase (HRP), diluted in blocking serum, for 1-3 hours at RT, shaking. A common dilution of secondary Ab is 1:20,000-50,000. Remember to confirm what the primary Ab was made in so that you use the correct secondary Ab.
  6. Wash blot 5X for 5 minutes in TBST at RT, shaking
  7. Process blot using SuperSignal® or Amersham's ECL detection kits.

Recipes

Regular (30:0.8) Laemlli SDS-PAGE Resolving Gel

 

7%

8%

10%

12%

15%

30:0.8 Acrylamide:Bis (ml)

5.1

5.87

7.33

8.81

11

1M Tris, pH 8.8 (ml)

8.24

8.24

8.24

8.24

8.24

10% SDS (ml)

0.2

0.2

0.2

0.2

0.2

50% Glycerol (ml)

1.12

1.12

1.12

1.12

1.12

H2O (ml)

7.3

6.53

5.11

3.59

1.4

10% Ammonium Persulfate (ml)

70

70

70

70

70

TEMED (ml)

7

7

7

7

7


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