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    GST pull down assay

    Xuefeng Ren, minghao chen

    Published: 2023-10-17 DOI: 10.17504/protocols.io.36wgqj2xxvk5/v1
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    Abstract

    GST pull down assay

    Steps

    1.

    Mix the GST tagged protein and fluorescent protein with 30 μl Glutathione Sepharose beads (Cytiva) at final 1 μM concentration in the buffer of 25 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 1 mM TCEP. The total volume is 200 μl.

    2.

    Rocking at 4°C

    3.

    Wash the beads four times, then eluted in 50 μl of buffer with 25 mM glutathione

    4.

    Mix 18 μl eluent in lithium dodecylsulfate (LDS)/BME buffer and subjected to SDS/PAGE gel without heating samples. The gel was scanned at 488 or 550 nm in ChemiDoc MP imaging system (Bio-Rad).

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