Microbiology

Applications of Chimeric Genes and Hybrid Proteins, Part B: by Melvin I. Simon

By Melvin I. Simon

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Additional resources for Applications of Chimeric Genes and Hybrid Proteins, Part B: Cell Biology and Physiology

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This allows the AP tag to be fused to the position where the native protein would enter the cell membrane, making it unlikely that the tag will interfere sterically with ligand binding. We generally position the fusion site immediately outside the hydrophobic transmembrane domain. The APtag-1, -2, and -5 vectors can be used for this purpose. The secretion signal sequence of the inserted protein is generally used, although with APtag-5 the signal sequence in the vector can be used instead. For proteins that are not membrane anchored in their native state, such as soluble ligands, we generally suggest making both a fusion to the N terminus of AP (with APtag-1, -2, or -5) and a fusion to the C terminus of AP (with APtag-4 or -5).

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Note that in experiments in which the desire is to coimmunoprecipitate other proteins that interact with the tagged protein, it may be necessary to omit the detergent from the precipitation buffer or consider alternative detergents so that protein interactions are not disrupted. Affinity Purifications with Epitope Tag Antibodies Protocol Two protocols are provided. The first is appropriate for small-scale immunopurification efforts performed in a microcentrifuge tube. 1. 5 M NaCl] at a 1 : 5 dilution in a microcentrifuge tube.

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