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:
- Cut a piece of nitrocellulose 5 cm x 5 cm or 10 cm by 10 cm if you have more samples.
- 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.
- 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.
- 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.
- Dot 2-5 ul of your desired proteins, peptides, antibodies, etc. using a pipette P20.
- Let dry spots. Can be left overnight in a petri dish.
- 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.)
- Rinse twice with TBST 1 minute. every.
- The primary antibody incubation: 10 mL total – the primary antibody at appropriate concentrations diluted in TBST + 1% BSA 1 hour
- Rinse twice with TBST 1 minute. every.
- 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
- Rinse twice with TBST 1 minute. every.
- 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
- Mix equal amounts of solution A and B of ECL (~ each A & B 1.5ml)
- stone manually abolished in the solution for 1 minute.
- around spotting wrap Saran Wrap
- take off the gloves when handling Film
- 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
- Place the film on top of the blot in the film cassette, make a note of the orientation of the film
- expose to 15sec -2 minutes, and if necessary, use another film and expose for anther 20 minutes.
- develop blot