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        Western Blotting

        互联网

        4411

        This protocol was developed for the BIORAD protein gel and transfer apparatus. The buffers can be used with any electrophoresis/transfer system.

        Solutions

        25 mM Tris 3.03 g Tris base

        192 mM glycine 14.4 g glycine

        20% MeOH 200 ml methanol

        up to 1 liter with Q

        do not adjust the pH it should be 8.3

        10X TBS

        100 mM Tris 8.0 100 ml 1 M Tris pH 8.0

        1.5 M NaCl 87 g NaCl

        up to 1 liter with Q

        to make TBST add Tween 20 to 0.1%

        to make prehybridization solution add serum to TBST to 5%

        Ponceau

        mix approximately 0.5 g Ponceau-S in 500 ml 1% acetic acid

        (Fisher# BP103-50)

        Developing Solution

        100 mM Tris 9.5 15 ml 1M Tris 9.5

        100 mM NaCl 3 ml 5M NaCl

        5 mM MgCl2 0.75 ml 1M MgCl2

        up to 150ml with Q

        To use, add 66 ml NBT and 50 ml BCIP to 15 ml of Developing Solution.

        NBT (sigma N-6876) 75 mg/ml in DMF, and BCIP (sigma B-6777 (pToluidine Salt) 50 mg/ml in DMF. Store at -20℃.

        Procedure

        • Run a standard SDS-PAGE gel and soak the gel in Transfer Buffer--30 minutes for 0.75 mm gels and 1 hour for 1.5 mm gels. Soak the nitrocellulose along with the gel.

        • Set up the transfer to nitrocellulose in 1X Transfer Buffer and transfer for 1 hour at 100 V or overnight at 30 V.

        • Remove membrane and soak in Ponceau for 5 minutes. Destain in 1% acetic acid and mark the molecular weight markers. Do not allow membrane to dry out.

        • Incubate membrane in TBST + 5% serum for several hours to overnight at room temperature. Alternately, the membrane can be incubated in PBS containing 5% Nonfat Dry Milk at 37° for 1 hour.

        • Add the primary antibody in 10 ml TBST + 5% serum and incubate for several hours. If using Milk Block, add primary antibody in 1X PBS and perform all washes in 1X PBS.

        • Wash 3X with TBST + 5% serum and add the secondary antibody in 10 ml TBST + 5% serum.

        • Wash 3X with TBST.

        • Add the developer solution and stop the reaction with 0.1 mM EDTA.

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