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Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation.

ABSTRACT: BACKGROUND:Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde cross-linking can fix transient and weak protein interactions, thereby reducing the number of false negatives but producing great complexity. To reduce this complexity, immunoaffinity purification can Fish out complexes that include particular target proteins, but affinity-based co-purification has a limited capacity to eliminate nonspecific binding to beads and/or antibodies. To Filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification. RESULTS:We described a 4 F strategy to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation: Fast Fix, Fish, and Filter, using albumin interactome as an example. The use of gel excision without staining makes this strategy comprehensive and sensitive. The target protein must be identified in the same slice as its ligands. The ligands must be identified in slices for the experimental group but not in the corresponding control slices. Only proteins that appear in the range of molecular weights equal to or greater than the sum of the proteins' theoretical molecular weights, together with the target, are considered ligands. In this study, 5 s of cross-linking with 10% formaldehyde was achieved in human blood. The use of this strategy identified 35 ligands for albumin. Comparison with four major previous studies of the albuminome revealed that 68.57% of the 35 ligands identified in our study were identified in these other studies. CONCLUSIONS:Fast cross-linking was achieved. The 4 F strategy can be used to identify real-time in situ interactions without prior intervention and to comprehensively identify ligands of particular target proteins with fewer false positives.

SUBMITTER: Zhu L

PROVIDER: S-EPMC3922604 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation.

Zhu Lisi L Li Menglin M Wei Lilong L Liu Xuejiao X Yin Jianrui J Gao Youhe Y

Proteome science 20140201 1

<h4>Background</h4>Fast Fixation is necessary to study real-time protein-protein interactions under physiological conditions. Fast formaldehyde cross-linking can fix transient and weak protein interactions, thereby reducing the number of false negatives but producing great complexity. To reduce this complexity, immunoaffinity purification can Fish out complexes that include particular target proteins, but affinity-based co-purification has a limited capacity to eliminate nonspecific binding to b ...[more]

PMID: 24484773

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Project description:Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

Dataset Information

Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation.

Publications

Fast fixing and comprehensive identification to help improve real-time ligands discovery based on formaldehyde crosslinking, immunoprecipitation and SDS-PAGE separation.

Similar Datasets

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