Western Dot Blot Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Western Dot Blot Protocol


20 mM Tris, pH 7.4
150 mM NaCl

TBS Tween—TBST (for washing blots)
Tween-20 (Brown bottle) +1mL by pipetting it in with pipettor or plastic bulb
stir half an hour

Blotting protocol:

1. Cut a piece of nitrocellulose 5 cm by 5 cm or 10 cm by 10 cm if you have more samples.
2. Remove the top layer of paper on top of the nitrocellulose.  Using a pencil and ruler, measure and draw out grids 1 cm by 1cm on the nitrocellulose.  Always wear gloves when handling the nitrocellulose.
3. Put the top layer of paper back on top of the nitrocellulose.  Cut off the top left corner of the nitrocellulose so that you have a frame of reference.
4. Using tweezers to help, remove the two piece of papers on top and below the piece of nitrocellulose, and place the nitrocellulose blot face up in a petri dish for dotting.
5. Dot 2-5 ul of a your desired protein, peptide, antibody, etc. using a P20 pipet.
6. Allow the dots to dry.  Can leave overnight in the petri dish.
7. Block with 5% skim milk (made in TBST) for 1hour. (some proteins require a higher percentage of skim milk e.g. 5% and incubation in the cold room for longer e.g. 30min.)
8. Rinse twice with TBST 1 min. each.
9. Primary antibody incubation: 10mL total--Primary antibody diluted to the appropriate concentration in TBST + 1% BSA 1hr
10. Rinse twice with TBST 1 min. each.
11. Secondary antibody incubation: 1:5000 dilution of the horseradish peroxidase secondary (choose the right secondary—either goat anti-rabbit horseradish peroxidase (HRP), or goat-anti mouse HRP depending on the type of primary antibody used), 30 minutes
12. Rinse twice with TBST 1 min. each.
13. Rinse twice with TBS + 1% Triton X-100 30 min. each, then develop the blot.
(Note that horseradish peroxidase is an enzyme and thus temperature sensitive, so it is best to leave it at 4 degrees if you can not immediately continue to develop it; however, try not to leave it at 4 degrees for too long [1-2 hours is probably OK])

Viewing the blot
1. mix equal amounts of solution A & B of ECL (~1.5mL each of A & B)
2. rock blot in that solution manually for 1 min. 
3. wrap Saran Wrap around blots
4. take off gloves when handling film
5. turn off all lights (can leave a dim red light on) when removing film from the box, and place remaining film back into box before dealing with the newly removed piece of film
6. place film on top of blot in the film cassette, making a note of the orientation of the film 
7. expose for 15sec -2 minutes, and if required, use another film and expose for anther 20 min.
8. develop the blot

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