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I. Prepare plant tissue samples: 1. Weigh ~0.5g fresh leaf tissue (3-5cm2), grind using liquid nitrogen. 2. Add ~0.4ml Plant Extraction Buffer, grind until slurry, transfer to 1.5ml tube. 3. Spin down for 15 minutes at 4°C, transfer 0.2-0.3 ml supernatant to fresh tube. 4. Add an equal volume of 2X Laemmli's buffer, boil for 5 min. 5. Use immediately to load gel or store at -20°C (reboil before loading gel). II. Pour Acrylamide Gel:
Mix and pour gel, then layer on top with butanol; allow to polyermize (30 min.), rinse off the butanol with H2O before layering on Stacking Gel.
Insert comb, allow to polymerize (30 minutes); remove comb just before loading samples. III. Gel Electrophoresis: 1. Setup gel in electrophoresis apparatus, fill reservoirs with 1X Running Buffer, check to remove air bubble from between plates at bottom of gel. 2. Remove comb and rinse wells with 1X Running Buffer. 3. Boil samples and standards for 5 min., then load gel (15-25µl for Hoefer mini-gel, 20-50µl for midi-gel). 4. Run until BPB-dye reaches bottom of gel (about 1-1.5hr for mini-gel at 40mA, about 2-2.5 hours for midi-gel at 200V). 5. If staining protein, cut away stacking gel and place running gel in Coomassie Blue Stain. 6. Destaining: Decant off stain and soak gel in Destain I Transfer gel into Destain II until background removed Observe on light box Vacuum dry for permanent record and/or densitometric analysis. IV. Gel to Filter Blotting: 1. Remove gel from plate-sandwich (note L to R orientation), cut away stacking gel and agar plug (if use), rinse running gel briefly in semi-dryTransfer Buffer (sdTB): 2. Cut six Whatman filter papers and one Immobilon-P filter to the size of the gel, soak Immobilon-P filter in methanol for 5 seconds, and then wet all filters with sdTB. 3. a) Lay 2-gel plastic manifold in Hoefer Electro-blotter. &nsbp; b) Lay 2 wetted filter papers for each gel onto platform of Blotter. &nsbp; c) Place Immobilon-P filter onto filters (need to mark or cut notch on desired corner of filter to maintain gel-lane orientations). d) Place gel in known orientation onto Immunobilon-P filter. e) Place 3 more wetted filters on top of stack, removing bubbles by rolling pipet. f) Install top of Electro-blotter and run at 150mA for 30-40 minutes (maximum 1 hour). 4. Remove Immunobilon filter(s), rinse with TBSt, and mark MW bands andgel top (T) with lab marker or pencil. 5. Can begin western probing immediately or store filter, either a) rinse in TBSt, place in seal-bag and store at 4°C, or b) place in Blocking Solution in seal-bag and store at -20°C. Note: Be aware of what type of membrane you are using as it could affect how you want to store. V. Probing the Filter: 1. Rinse filter in TBSt for ~5 min, then place in seal-bag with 10-20ml Blocking Solution for at least one hour at room temperature: Blocking Solution: 10-20% goat serum albumin 5% Carnation non-fat dry milk in TBSt buffer 2. Add Primary Antibody (1µg/ml) directly to Blocking Solution in seal-bag, incubate at room temperature for 1-2 hours. 3. Transfer filter to small dish and wash 3X 5 minutes with TBSt. 4. Place filter in seal-bag with 10-20 ml Blocking Solution and add Secondary Antibody to the dilution of 1:2000 (probably AP-labelled, sheep anti-mouse IgG), incubate at room temperature for ~30 minutes. 5. Wash filters with two quick rinses, then 3X 5 minutes with TBSt. 6. To develop with ECL Chemifluorescent Kit: -mix equal parts of ECL solutions A and b (~1 ml each per filter) -spread ECL Solution Mix onto protein side of filter and incubate for 1 minute -drain filter and wrap in plastic wrap, carry to darkroom in film cassette, and expose to ECL Hyperfilm. Exposure times range from 30 sec to 10 min. Western Blot Reagents:
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