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    Protein Lysate Preparation Protocol (Tissue)

    Scott Vermilyea

    Published: 2024-03-21 DOI: 10.17504/protocols.io.n2bvj3k3xlk5/v1
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    Abstract

    This protocol details the preparation of protein lysate of the tissue sample.

    Attachments

    Western Blot and Lysate Prep Protocol.docx

    Steps

    Protein Lysate Preparation (Tissue)

    1.

    If tissue stored at -80°C, place in -20°C for at least 4h 0m 0s or prior to homogenization.

    2.

    Weigh tissue out in mg.

    3.

    Add in 10 volumes of 1X TNE (e.g. 10µL of TNE per 1mgof tissue).

    4.

    Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and On ice.

    5.

    This is TNE crude lysate (no detergents).

    6.

    Add equal volume of 1xTNE +1% SDS, 0.5% NP-40, and 0.5% Doc (Complete TNE).

    7.

    Sonicate at 40°C (3 pulses: 10s On/2s Off at 20% amplitude).

    8.

    Boil (0h 10m 0s).

    Note
    STOPPING Point: Sample should be in “Complete TNE”. Make sure to sonicate and boil prior storage.

    9.

    Centrifuge (16000x g,4°C).

    10.

    Collect supernatant for BCA protein assay.

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