Western Blot Analysis of HsRAD52-FLAG Protein

Wild-type and mutant HsRAD52-FLAG proteins were detected using Western blot analysis as described previously [29 (link)]. Whole cell extracts of cells of the appropriate genotype were prepared by glass bead disruption, and the proteins separated on acrylamide gels before transfer to nylon membranes (Imobilon-P PVDF, Millipore Sigma, St. Louis, MO, US). Proteins were detected with anti-FLAG M2 (Millipore Sigma) and anti-GAPDH (Aviva Systems Biology, San Diego, CA, USA) primary antibodies, goat anti-mouse HRP-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA), chemiluminescent signal generation (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific), and visualization on X-ray film.

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Clear A.D., Manthey G.M., Lewis O., Lopez I.Y., Rico R., Owens S., Negritto M.C., Wolf E.W., Xu J., Kenjić N., Perry J.J., Adamson A.W., Neuhausen S.L, & Bailis A.M. (2020). Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast. Microbial Cell, 7(10), 270-285.

Publication 2020

anti-FLAG M2 goat anti-mouse HRP-conjugated secondary antibody SuperSignal West Femto Maximum Sensitivity Substrate

Acrylamide Anti antibody Antibodies Cell Cells extracts Gapdh Gels Genotype Goat Membranes Mouse Mutant proteins Nylon Proteins Pvdf Sensitivity Western blot analysis X ray film

Corresponding Organization : Thomas Jefferson University

Other organizations : Arnold Ventures, California State Polytechnic University, University of California, Los Angeles, University of California, Davis, Pomona College, University of California, San Francisco, University of Pennsylvania, University of California, Riverside

Top 5 similar protocols

Variable analysis

independent variables

Wild-type HsRAD52-FLAG protein
Mutant HsRAD52-FLAG protein

dependent variables

Detection of wild-type and mutant HsRAD52-FLAG proteins using Western blot analysis

control variables

Whole cell extracts of cells of the appropriate genotype
Glass bead disruption for cell lysis
Acrylamide gel electrophoresis for protein separation
Transfer of proteins to nylon membranes (Imobilon-P PVDF)
Detection with anti-FLAG M2 and anti-GAPDH primary antibodies
Goat anti-mouse HRP-conjugated secondary antibody
Chemiluminescent signal generation (SuperSignal West Femto Maximum Sensitivity Substrate)
Visualization on X-ray film

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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