Western Blot Detection with Horseradish Peroxidase

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Western blot Detection with Horseradish Peroxidase

Recipes:

10x Phosphate Buffered Saline—PBS (for removing methanol from blots)
NaCl 80g (1.37 M for 10x)
KCl 2g (27mM for 10x)
Na₂HPO₄(80mM for 10x)
KH₂PO₄(20mM for 10x)
water to 1L
pH to 7.4 with HCl

PBS Tween—PBST (for washing blots)
PBS 2L
Tween-20 (Brown bottle) +1mL by pipetting it in with pipettor or plastic bulb
stir half an hour

Protocol :

Blotting
1. Wet a dried, transferred blot with methanol if the blot is made with PVDF, or soak in PBS if blot is made from nitrocellulose (note that methanol is toxic, so wear gloves and don’t inhale it) FOR NITROCELLULOSE BLOTS DON’T WET WITH METHANOL, OR ELSE IT’LL DISTINTEGRATE IN YOUR HANDS
2. Rinse ~4x with PBS to get rid of the methanol
3. Place the blot face up for blotting
4. Block with 1% skim milk (made in PBS) for 10 min. (some proteins require a higher percentage of skim milk e.g. 5% and incubation in the cold room for longer e.g. 30min.)
5. Primary antibody incubation: 10mL total--Primary antibody diluted to the appropriate concentration in PBS (+0.2% milk) 1hr
6. Washes: 3x 10min. each PBST
7. 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
8. More washes: ~5x PBST 5-10 minutes each
(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 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