Whole Cell Extract Preparation and Western Blot Analysis

Whole cell extracts were prepared using the TCA extraction method, essentially as described^{35 (link)}. Samples were analyzed by standard SDS-PAGE and Western blotting. Anti-c-myc monoclonal antibody (Roche; 11667149001) was diluted 1:1,000 to detect Rfc1-AID; Arp7 polyclonal antibody (Santa Cruz, SC-8961) was used at 1:5,000 dilution as a loading control. Secondary antibodies, used at 1:5,000 dilution, were IRDye® 800CW Donkey anti-Mouse IgG (LI-COR, 925–32212) and IRDye® 680LT Donkey anti-Goat IgG (LI-COR, 925–68024). Membranes were imaged with an Odyssey Infrared Imager (LI-COR). Quantification of protein bands was performed using Image Studio Lite 5.0.21 software.

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Kulkarni D.S., Owens S.N., Honda M., Ito M., Yang Y., Corrigan M.W., Chen L., Quan A.L, & Hunter N. (2020). PCNA activates the MutLγ endonuclease to promote meiotic crossing over. Nature, 586(7830), 623-627.

Publication 2020

Anti-c-myc monoclonal antibody Arp7 polyclonal antibody IRDye® 800CW Donkey anti-Mouse IgG IRDye® 680LT Donkey anti-Goat IgG Odyssey Infrared Imager

Anti c antibody Anti igg Antibodies Antibody Cell extracts Dilution Donkey Goat Irdye 800cw Membranes Mouse Protein Rfc1 Sds page

Corresponding Organization :

Other organizations : Howard Hughes Medical Institute, University of California, Davis

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Variable analysis

independent variables

TCA extraction method

dependent variables

Protein expression levels of Rfc1-AID and Arp7

control variables

Preparation of whole cell extracts
SDS-PAGE and Western blotting analysis
Use of anti-c-myc monoclonal antibody (1:1,000 dilution) to detect Rfc1-AID
Use of Arp7 polyclonal antibody (1:5,000 dilution) as a loading control
Use of secondary antibodies (1:5,000 dilution): IRDye® 800CW Donkey anti-Mouse IgG and IRDye® 680LT Donkey anti-Goat IgG
Odyssey Infrared Imager (LI-COR) for membrane imaging
Image Studio Lite 5.0.21 software for protein band quantification

controls

Arp7 polyclonal antibody (1:5,000 dilution) used as a loading control

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