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        【求助】EMSA抗体的选择

        丁香园论坛

        3620
        请教各位达达,我准备做EMSA,如果做SUPER-SHIFT的话,该选择原装抗体还是分装的抗体呢,盼不吝赐教!
        看到一篇EMSA的文章,不知道里面有没有回答你的问题哈,不过英文的值得阅读哈。
        GEL SUPER-SHIFT ASSAY/ EMSA
        Materials/Reagents:
        See Gel-shift assay
        Procedure:
        1. Prepare Whole Cell Extract. Make sure every solution DOES NOT contain DTT.
        2. Add 1 or 2 μl of non-diluted monoclonal antibody (4B4 is better than 10H8 for HSF1) or
        1/10 diluted polyclonal antibody to 10 μg of whole cell extract from HeLa in 5 μl of
        Buffer C; incubate on ice for 30 min. (Monoclonal antibody does not disturb HSF1-DNA
        binding activity.)
        Following steps are the same as normal gel shift:
        3. Add reaction mix containing 1-10 fmol (~0.1 ng) of 32P-labeled probe and 0.5 μg of
        poly(dI/dC) in 20 μl of 1X Binding buffer. Incubate at RT for 20 min.
        4. Add 2 μl of loading dye, load total amount of the sample on 4% 0.25X TBE gel which
        has been pre-run for at least 20 min in 0.25X TBE buffer.
        5. Electorophoresis with 100-130 V in 0.25X TBE buffer for 3 hours.
        You may use 18 cm-height gel system and stop running after dye front comes to 12 cm
        point from the top. When a gel was run with 130 V, the current was less than 20 mA. You
        could run a gel with 100V. It might give better separation.
        Troubleshooting/Critical Parameters:
        • The reaction mixture MUST NOT contain DTT, because DTT kills antibody.
        • Polyclonal antibody of HSF1 can be added after mixing of WCE and DNA probe. In that
        case, the antibody should be added in 1:100 to 1:500 final dilution.
        • For Hsp70, 3A3 and C92 did work, 4G4 and 5A5 did not. 3A3 may be used 1 μl/
        sample.
        • HSE probe must have high-specific activity (>10,000 cpm/fmol) and be double-stranded.
        • If you did not have good separation or running, you should newly prepare acrylamide and
        TBE stock solution.
        Thanks to Wanglinling!
        我这里有做EMSA的资料,留个邮箱我发给你

        本文由丁香园论坛提供,想了解更多有用的、有意思的前沿资讯以及酷炫的实验方法的你,都可以成为师兄的好伙伴

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        【求助】EMSA抗体的选择

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