Western Blot Analysis of DNA Repair Proteins

Anti-XRCC4 rabbit polyclonal antibody [47 (link)], anti-FLAG mouse monoclonal antibody (clone M2; F3165; Sigma-Aldrich; St. Louis, MO, USA), anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mouse monoclonal antibody (clone 6C5; MAB374;) and anti-LIG4 guinea pig polyclonal antibody (gifted by Prof. Miki Shinohara, Kinki University) [36 (link)] were used as the primary antibody at 1/1000 to 1/5000 dilution. As the secondary antibody, horseradish peroxidase-conjugated anti-rabbit immunoglobulins swine polyclonal antibody (P0399; Dako; Glostrup, Denmark), anti-mouse immunoglobulins goat polyclonal antibody (P0447; Dako) or anti-guinea pig immunoglobulins rabbit polyclonal antibody (P0141; Dako) was used at 1/1000 to 1/3000 dilution. The immunocomplexes were developed using WesternSure Chemiluminescent Western Blot Reagent (LI-COR; Lincoln, NE, USA) and the images were captured by C-Digit Blot Scanner (LI-COR). Protein Ladder One Plus, Triple-color (Nacalai Tesque) was used as the molecular weight standard. Other procedures of western blotting followed earlier publications [31 , 32 (link)].

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Asa A.D., Wanotayan R., Sharma M.K., Tsukada K., Shimada M, & Matsumoto Y. (2021). Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity. Journal of Radiation Research, 62(3), 380-389.

Publication 2021

anti-FLAG mouse monoclonal antibody (clone M2; P0141 WesternSure Chemiluminescent Western Blot Reagent C-Digit Blot Scanner

Anti immunoglobulins Antibody Clone Digit Dilution Glyceraldehyde 3 phosphate dehydrogenase Goat Guinea pig Horseradish peroxidase Lig4 Monoclonal antibody Mouse Protein Rabbit Swine Xrcc4

Corresponding Organization : Tokyo Institute of Technology

Other organizations : Mahidol University

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Variable analysis

independent variables

Anti-XRCC4 rabbit polyclonal antibody [47 (link)]
Anti-FLAG mouse monoclonal antibody (clone M2; F3165; Sigma-Aldrich; St. Louis, MO, USA)
Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mouse monoclonal antibody (clone 6C5; MAB374;)
Anti-LIG4 guinea pig polyclonal antibody (gifted by Prof. Miki Shinohara, Kinki University) [36 (link)]

dependent variables

Protein expression levels measured by Western blotting

control variables

Antibody dilutions (1/1000 to 1/5000 for primary antibodies, 1/1000 to 1/3000 for secondary antibodies)
Protein Ladder One Plus, Triple-color (Nacalai Tesque) used as the molecular weight standard
Other Western blotting procedures followed earlier publications [31 (no link found), 32 (link)]

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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