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Western Blot Protocol

 

Put blot into antibody:

  1. Remove blot from fridge.
  2. Prepare primary antibody:
  3. 10mls TBS
  4. 2% Non-fat powdered milk
  5. 0.05% Tween
  6. Add appropriate amount of antibody for dilution (1:5,000, 1:1,000, 1:500, etc.).
  7. Prepare plastic bag:
    1. Cut plastic tube to size of gel.
    2. Seal bag with bag sealer set on 3.
    3. Put membrane blot in bag.
    4. Seal three sides of the plastic bag and cut excess (leave extra room on the top for when adding the antibody).
    5. Add antibody with pipette and aide.
    6. Completely wet membrane.
    7. Use a kimwipe to push out all the air bubbles.
    8. Seal bag closed once all bubbles are removed and cut bag excess.
    9. Put blot on rocker 1-2 hours at room temperature or overnight at 4 degrees Celsius.
    10. Take blot out of the bag and put in TBST rinse.
    11. TBST (For 1 Liter):
      1. 900mls H2O
      2. 100mls 10X TBS
      3. 0.5mls Tween
      4. Save primary antibody.  Store at 4 degrees Celsius.
      5. Put blot in tray with 100-200mls TBST and put on rocker for 3-5 minutes.
      6. Repeat rinse in TBST.
      7. Prepare secondary antibody (1:5,000):
        1. 25mls TBST
        2. 5ul secondary antibody
        3. Pour secondary antibody onto blot and put on rocker for at least 1 hour.
        4. Wash blot with 3 washes of TBST for 3-5 minutes each.
        5. Prepare Western Blotting Detection Solution:
          1. 3mls solution A
          2. 3mls solution B
          3. Lay glass plate down on bench and lay blot on glass plate.
          4. Put developer drop-wise over blots surface.
          5. Tilt blot to make sure the whole blot is covered.
          6. Let sit 1 minute.
          7. Lay out a piece of smooth seran wrap.
          8. Take blot off plate and let developer drip off.
          9. Lay blot protein side down onto seran wrap and fold seran wrap to seal blot in bag.
          10. Tape blot into cassette.
          11. Take Biomax light film, cassette, timer and scissors down to the dark room.
          12. Cut film into 2 pieces (fold upper right corner).
          13. Put film over blot and close cassette.
          14. Expose blot for 1 minute.
          15. Put film in developer.
          16. Expose blot for longer or shorter times depending on first exposure.
          17. Label film using blot (when it is still taped into the cassette).
          18. If using another antibody strip blot.

 

Strip blot:

  1. Rinse blot in TBST while preparing bag.
  2. Prepare bag by sealing three sides.
  3. Put blot in bag and add 50mls strip.
  4. Strip (For 50mls):
  5. 5mls 20% SDS
  6. 3.15mls 1M Tris pH 6.7
  7. 343ul B-Me
  8. 41.5mls H2O
    1. Remove bubbles using a kimwipe and seal bag.
    2. Put in 50 degree Celsius water bath for 30 minutes.
    3. Every 10-15 minutes shake bag.
    4. Remove from water bath and pour off strip (save strip at 4 degrees Celsius).
    5. Rinse blot in TBST for 1 minute.
    6. Rinse blot 3 times in fresh TBST for 3-5 minutes each on rocker (repeat rinse until the smell is gone).
    7. Put blot in MeOH for 30 seconds.
    8. Let blot air dry for 15 minutes.
    9. Store at 4 degrees Celsius or put in new antibody.