Western Blot Analyses: Detailed Antibody Protocols

Western blot analyses followed our standard protocols (11 (link), 44 (link), 45 (link), 46 (link)). The primary antibodies that were used are: α-RAD51AP1 (NB100-1129; Novus; 1:5000; and our own α-RAD51AP1 antibody, as previously described in (9 (link))), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:4000), α-ß-Actin (ab6276; Abcam; 1:3000), α-H3 (ab1791; Abcam; 1:10,000), α-H2A (GTX1129418; GeneTex; 1:1000); α-FLAG (F3165; Sigma; 1:1000); α-MBP (PAI-989; ThermoScientific; 1:5000). HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; 1:10,000) were used as secondary antibodies and SuperSignal Substrate kit (Thermo Scientific) for the detection of signal.

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Pires E., Sharma N., Selemenakis P., Wu B., Huang Y., Alimbetov D.S., Zhao W, & Wiese C. (2021). RAD51AP1 mediates RAD51 activity through nucleosome interaction. The Journal of Biological Chemistry, 297(1), 100844.

Publication 2021

NB100-1129 ab6276 ab1791 α-FLAG HRP-conjugated goat anti-rabbit or goat anti-mouse IgG

Actin Anti igg Antibodies Antibody Goat Mouse Novus Rabbit Signal detection Western blot analyses

Corresponding Organization : Colorado State University

Other organizations : The University of Texas at San Antonio, The University of Texas Health Science Center at San Antonio

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Variable analysis

independent variables

Primary antibodies used: α-RAD51AP1 (NB100-1129; Novus; 1:5000; and our own α-RAD51AP1 antibody), α-RAD54 (F-11; sc-374598; Santa Cruz Biotechnology; 1:500), α-RAD51 (Ab-1; EMD Millipore; 1:4000), α-ß-Actin (ab6276; Abcam; 1:3000), α-H3 (ab1791; Abcam; 1:10,000), α-H2A (GTX1129418; GeneTex; 1:1000), α-FLAG (F3165; Sigma; 1:1000), α-MBP (PAI-989; ThermoScientific; 1:5000)

dependent variables

Protein expression levels measured using Western blot analyses

control variables

Standard protocols for Western blot analyses (11, 44, 45, 46)
HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; 1:10,000) used as secondary antibodies
SuperSignal Substrate kit (Thermo Scientific) used for signal detection

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

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