Western Dot Blot Protocol

TBS
20 mM Tris, pH 7.4
150 mM NaCl

TBS TweenTBST (for washing blots)
TBS 2L
Tween-20 (Brown bottle) + 1mL by Pipetting in the pipettor or plastic balls
stir half an hour

Blotting protocols:

  1. Cut a piece of nitrocellulose 5 cm x 5 cm or 10 cm by 10 cm if you have more samples.
  2. Remove the top layer of paper on top of nitrocellulose. Using a pencil and a ruler, measure and draw out a grid of 1 cm by 1 cm in nitrocellulose. Always wear gloves when handling the nitrocellulose.
  3. Put back the top layer of paper on top of nitrocellulose. Cut off the top left corner of the nitrocellulose so you have a frame of reference.
  4. Using a pair of tweezers to help, remove two pieces of paper at the top and bottom pieces of nitrocellulose, and the nitrocellulose blot up in the petri dish to dotting.
  5. Dot 2-5 ul of your desired proteins, peptides, antibodies, etc. using a pipette P20.
  6. Let dry spots. Can be left overnight in a petri dish.
  7. Block with 5% skim milk (made in TBST) for 1 hour. (Some proteins require a higher percentage of say 5% skim milk and incubation in a cold room for longer eg 30 mins.)
  8. Rinse twice with TBST 1 minute. every.
  9. The primary antibody incubation: 10 mL total – the primary antibody at appropriate concentrations diluted in TBST + 1% BSA 1 hour
  10. Rinse twice with TBST 1 minute. every.
  11. Secondary antibody incubation: 1: 5000 dilution of horseradish peroxidase secondary (right pick secondaryeither goat anti-rabbit horseradish peroxidase (HRP), or goat anti-mouse HRP antibody depends on the type of primer used), 30 minutes
  12. Rinse twice with TBST 1 minute. every.
  13. Rinse twice with TBS + 1% Triton X-100 30 minutes. respectively, and then develop the blot.
    (Note that the horseradish peroxidase enzyme and thus are sensitive to temperature, so it is best to leave it at 4 degrees if you can not immediately continue to develop it, but try not to leave it at 4 degrees too long [1-2 hours probably OK])

Seeing blot

  1. Mix equal amounts of solution A and B of ECL (~ each A & B 1.5ml)
  2. stone manually abolished in the solution for 1 minute.
  3. around spotting wrap Saran Wrap
  4. take off the gloves when handling Film
  5. Turn off all the lights (can leave a dim red light on) when removing the film from the box, and place the remaining film back into the box before dealing with a new piece of deleted movie
  6. Place the film on top of the blot in the film cassette, make a note of the orientation of the film
  7. expose to 15sec -2 minutes, and if necessary, use another film and expose for anther 20 minutes.
  8. develop blot

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