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        植物叶蛋白western blotting

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        3453
        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:

        Running gel, lower (10%)(~30 mls for 2 gels:
        Acrylamide (40%)
        dH2 O
        1M Tris (pH 8.7)
        10% SDS
        10% APS (Ammonium persulfate)
        TEMED
         
        7.5ml
        11.0ml
        11.2ml
        300µL
        120µL
        20µL


        Mix and pour gel, then layer on top with butanol; allow to polyermize (30 min.), rinse off the butanol with H2 O before layering on Stacking Gel.

        Stacking gel, upper (for 2 gels): Acrylamide (40%)
        dH2 O
        1M Tris-HCl (pH 6.8)
        10% SDS
        10% APS
        TEMED
         
        1.3ml
        7.25ml
        1.3ml
        104µl
        52µl
        11µl


        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:
        Plant Extaction Buffer (final concentration):
        10mM NaCl
        10mM MgCl2
        5mM EDTA
        10mM β-mercaptoethanol
        25mM Tris-HCl (pH 7.5)
        1mM PMSF


        Laemmli's Sample Buffer (2X):
        1.0M Tris-HCl (pH 6.8)
        10% SDS
        glycerol (37.7g)
        β-mercaptoethanol
        bromophenol blue
        -raise to 100ml with dH2 O
        12.5ml
        40ml
        ~30ml
        1ml
        "a pinch"


        Laemmli's Running Buffer (10X, dilute to 1X for PAGE):
        -start with approx. 600ml dH2 O, add:
        Trizma Base
        glycine
        10% SDS
        -raise to 1L with dH2 O
         


        30.28g
        144.14g
        100ml


        Coomassie Blue Stain (for 500ml):
        glacial acetic acid
        methanol
        -raise to 500ml with dH2 O, add 2g Coomassie blue G-250
         

        100 ml
        250ml


        Destain I (for 1L):
        methanol
        glacial acetic acid
        -raise to 1L with dH2 O
         
        500ml
        100ml


        Destain II (for 1L):
        methanol
        glacial acetic acid
        -raise to 1L with dH2 O
         
        50ml
        70ml


        TBS (for 1L):
        1.0M Tris-HCl, pH 7.5
        5.0M NaCl
        -raise to 1L with dH2 O, adjust pH to 7.5
         
        10ml
        30ml


        TBSt (for 4L):
        -start with approx. 3L dH2 O, add:
        Trizma Base
        NaCl
        -raise to 4L with dH2 O, adjust pH to 7.5, then add ~4ml Tween 20


        9.6g
        32g


        semi-dry Transfer Buffer (sdTB, for 400ml):

        -start with approx. 250ml dH2 O, add:
        Trizma Base
        glycine
        methanol
        -raise to 400ml with dH2 O
         


        1.2g
        5.66g
        80ml


        Blocking Solution (make fresh, for 50ml):

        -into clean, 50ml conical tube, weigh: Carnation non-fat, dry milk
        -dissolve into approx. 30ml TBSt, add: Goat Serum Albumin
        -raise to 50ml with TBSt

        2.5g (5%)
        5-10ml (10-20%)
        <center> <p>  </p> </center>
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