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Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
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Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

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Immunoprecipitation: lysing bacteria by sonication

Ed Harlow, David Lane

    CSH Protocols
    |April 10, 2012
    PubMed

    Abstract:

    INTRODUCTIONIn this protocol, bacterial cells are lysed by being subjected to short, intense treatments with ultrasound, which breaks the cell walls and shears the DNA into sizes that will not affect the viscosity of the samples. Note that this method causes some denaturation of the samples. The resulting lysate is ready for preclearing.

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    Immunoprecipitation01:20

    Immunoprecipitation

    Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
    Chromatin Immunoprecipitation
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    Tissue Homogenization and Cell Lysis01:32

    Tissue Homogenization and Cell Lysis

    Tissue homogenization involves disintegrating tissue architecture and lysing cells, and is an early step in isolating and analyzing cellular components. The method used for homogenization depends on the sample type, the amount of sample available, the analyte to be obtained, and the sensitivity of the method. These methods are broadly classified as mechanical and non-mechanical methods.
    Mechanical methods of tissue homogenization
    These methods rely on applying external physical force to disrupt...

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