Co-immunoprecipitation of HA-tagged EspZ

Co-immunoprecipitation studies were performed using transiently transfected HeLa cells expressing HA-tagged EspZ. At 72-hours post-transfection, cells were harvested via centrifugation, and proteins were extracted using IP Lysis/Wash Buffer Supplemented with 1% CHAPs (Thermo Fisher Scientific).^{30 (link),37 (link)} Immunoprecipitation was performed on 2 mg protein extracts using α-FIS1 antibody (Santa Cruz Biotechnology), α-HA antibody (Millipore Sigma) and Pierce Crosslink IP Kit (Thermo Fisher Scientific) following manufacturer’s protocol. Immunoprecipitated samples were resolved via a 4–20% SDS-PAGE. The presence of HA-tagged EspZ and FIS1 was confirmed by western blot using α-HA and α-FIS1 antisera (Millipore Sigma).

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Roxas J.L., Ramamurthy S., Cocchi K., Rutins I., Harishankar A., Agellon A., Wilbur J.S., Sylejmani G., Vedantam G, & Viswanathan V.K. (2022). Enteropathogenic Escherichia coli regulates host-cell mitochondrial morphology. Gut Microbes, 14(1), 2143224.

Publication 2022

CHAPs Pierce Crosslink IP Kit

Antibody Antisera Buffer Cells Centrifugation Chaps Co immunoprecipitation Hela cells Immunoprecipitation Protein Sds page Transfection Western blot

Corresponding Organization : University of Arizona

Other organizations : Southern Arizona VA Health Care System

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Variable analysis

independent variables

Transient transfection of HeLa cells with HA-tagged EspZ

dependent variables

Immunoprecipitation of proteins using α-FIS1 and α-HA antibodies
Detection of HA-tagged EspZ and FIS1 proteins by Western blot

control variables

Protein extraction using IP Lysis/Wash Buffer supplemented with 1% CHAPs
Use of Pierce Crosslink IP Kit for immunoprecipitation according to manufacturer's protocol

controls

Positive control: Immunoprecipitation using α-HA antibody to detect HA-tagged EspZ
Negative control: Not explicitly mentioned

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