Comprehensive Antibody Immunostaining Protocol

Antibodies are rabbit polyclonal or mouse monoclonal (mAb), unless specified. From Covance: mAb anti-HA conjugated to fluorescein-isothiocyanate (cat# FITC-101L; 1:50 dilution) and mAb anti-HA (#MMS-101R; 1:500 IB and 1:100 IF); from BD Biosciences: mAb anti-Orc4 (#611171; 1:500); from BD Transductions: mAb anti-Cdk2 (#610146; 1:500); from Cell Signaling: mAb anti-Histone H4 (#2935; 1:500), anti-Mcm3 (#4012; 1:200), anti-Mcm4 (#12973; 1:500), anti-Smad2 (#5339; 1:1000), and rabbit monoclonal anti-Smad3 (#9523; 1:200); from Invitrogen: anti-BrdU-Alexa488 (#B35130; 1:20) and anti-BrdU-Alexa594 (#B35132; 1:20); from Calbiochem: anti-Rb (#PC13 ; 1:500); from Millipore: rabbit monoclonal anti-Smad4 (#04–1033; 1:750); from Santa Cruz Biotechnology: mAb anti-Mcm6 (#sc-55577; 1:200), mAb anti-Mcm7 (#sc-9966; 1:1000), anti-Cdc6 (#sc-8341; 1:300), anti-Cdc6-serine54P (#sc-12920R; 1:500), anti-CycE (agarose-conjugated; #sc-481; 1:20), mAb anti-Cyclin E (#sc-377100; 1:100),and anti-Cdt1 (#sc-28262; 1:300); from Sigma: mAb anti-Actin (#A5316; 1:5000); anti-Mcm2 (1:1000) was generated by our group and validated previously (27 (link)); anti-Mcm5 (1:2000) was provided by Rolf Knippers (University of Konstanz, Germany); anti-Myc (1:3000) was provided by Steve Hann (Vanderbilt University); anti-HBO1 (1:200) was provided by Masayoshi Iizuka (Teikyo University School of Medicine).

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Nepon-Sixt B.S, & Alexandrow M.G. (2018). TGFß1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-deficient Conditions. Molecular cancer research : MCR, 17(1), 277-288.

Publication 2018

mAb anti-HA anti-Smad2 anti-Smad3 anti-Smad4 mAb anti-Actin

Actin Agarose Alexa594 Antibodies Brdu Cdk2 Cyclin e Dilution Fitc Fluorescein Histone h4 Isothiocyanate Mcm2 Mcm4 Mcm5 Mcm6 Mouse Orc4 Rabbit Smad2 Smad3 Smad4

Corresponding Organization : Moffitt Cancer Center

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Variable analysis

independent variables

Antibody types (rabbit polyclonal or mouse monoclonal)
Antibody sources (Covance, BD Biosciences, BD Transductions, Cell Signaling, Invitrogen, Calbiochem, Millipore, Santa Cruz Biotechnology, Sigma, and custom-generated)

dependent variables

Not explicitly mentioned

control variables

Antibody dilutions (e.g., 1:50, 1:100, 1:200, 1:300, 1:500, 1:750, 1:1000, 1:3000, 1:5000)

controls

Positive control: anti-Mcm2 antibody generated by the authors and validated previously
Positive control: anti-Mcm5 antibody provided by Rolf Knippers
Positive control: anti-Myc antibody provided by Steve Hann
Positive control: anti-HBO1 antibody provided by Masayoshi Iizuka

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