Western Blot
From ChoanoWiki
WESTERN BLOT
Run protein on SDS-Page gel:
1. clean and assemble glass plates and gel casting station
2. fill gel casting setup ¼ full with methanol and mark level on plates; wait 10 minutes to determine if apparatus is leaking
3. decant methanol and dry with kim wipe
Mix SDS page solutions: (10% gel):
Resolving layer:
7.2 mL water
3.75 mL 40% acrylimide (1:29)
3.8 mL 1.5M Tris (pH 8.8)
0.15 mL 10% SDS
0.15 mL 10% APS *
6µl TEMED *
Stacking layer:
5.8 mL water
1.0 mL 40% acrylimide (1:29)
1.0 mL 1.5M Tris (pH 6.8)
0.08 mL 10% SDS
0.08 mL 10% APS *
8µl TEMED *
- Do not add until ready to pour each layer
Pour resolving layer:
1. add APS and TEMED, swirl to mix
2. pour 3.5mL gel into casting frame
3. overlay with a thin layer of methanol
4. let stand for 30 minutes
Pour stacking layer:
1. decant methanol from gel and wick off excess with kim wipes
2. add APS and TEMED, swirl to mix
3. fill casting station to top and slowly insert comb (use eye protection)
4. let stand 30 minutes
Assemble SDS Page tank:
1. remove gel plates from casters
2. place on electrode assembly (short plate towards the inner chamber
3. place electrode assembly in white clamping frame and place in buffer tank
4. fill inner and outer buffer chamber with 1x Running Buffer
5. remove bubbles from bottom of gel (usually not an issue)
6. remove comb and rinse wells with 1x running buffer (not important to rinse)
Sample prep:
1. mix sample with 4x loading buffer [containing 1.4µl ßmer:100µl loading buffer]. Can fit 200µl per in a single-well gel and 20µl per lane in a 10 well gel.
2. load 10µl per lane of RPN756 ladder
3. Run gel at less than 220V until dye front runs off completely
Transfer:
1. for each gel, cut to size and soak in 1x Transfer Buffer:
A. 1 nitrocellulose membrane (touch only with gloves)
B. 2 pieces of Whatman paper
C. 3 sponges
2. remove gel from buffer tank and gently separate plates with a razor
3. while submerged in 1x Transfer Buffer, make gel “sandwich”: layer on gray side of cassette - sponge → whatman → gel → membrane → sponge; Make sure that there are no air bubbles between layers
4. fold and clamp cassette, place in holder with gray side of the cassette near the gray side of the holder
5. place small stir bar and ice block inside tank and fill with 1x Transfer Buffer
6. place on stir plate in cold room (stir slowly); trnaser @ 0.35 Amps for 1 hour
- Option: visualize transferred protein
1. apply 1mL of Ponceaus Reagent to membrane
2. wash evenly until protein bands are visible
3. mark blot with pencil to allow re-alignment
4. cut membrane
5. wash until colorless with 1x PBS, 0.1% Tween
Blot:
1. incubate membrane in block O/N @ 4 C or 1 hr/room temp/shaking
2. incubate membrane with primary antibody in blocker 1hr/room temp/shaking or overnight at 4 C
3. wash in 1X PBS, 0.1% Tween:
-one quick wash
-2x 5 min wash/room temp/shaking
-one quick wash
4. incubate membrane with secondary antibody in blocker 1 hour/room temp/shaking
5. wash blot in 1x PBS, 0.1% Tween
-one quick wash
-1x 5 minutes wash/room temp/shaking
-1x 10 minutes wash/room temp/shaking
-one quick wash
Develop:
1. line film cassette with plastic wrap and tape it down so that it is smooth and will not move
2. place membrane in tip box lid @ an incline
3. add 1mL of each Pierce Super Signal reagent to the bottom of the tip-box lid and mix
4. constantly pipette over the membrane for 5 minutes and blot excess
5. place blot on plastic wrap in film box
6. align the ladder lane next to it
7. in dark room place film over membrane and expose 5 minutes, then develop. Adjust exposure time from 1 second to 1 hour, as needed.
1X transfer buffer:
3g Tris base
14.4g Glycine
200mL methanol
water to 1 L
Blocker [Make Fresh]:
(1x PBS; 5% Milk; 0.1% Tween)
20mL 20X PBS
20g powdered milk
0.4mL Tween
water to 400mL
10X running buffer:
30.4 g Tris base
154g Glycine
10g SDS
water to 1L
4x loading buffer:
1mL 1M Tris pH 6.8
4mL 10% SDS
4mL 100% glycerol
1mL water
dash of bromophenol blue
