For SDS-PAGE, samples were denatured with 100 mM dithiothreitol (DTT) and 1× Laemmli buffer. Proteins were fractionated on either 4 to 12% NuPAGE Novex Bis-Tris gels or 7% NuPAGE Tris-acetate gels (Thermo Scientific). Proteins were transferred onto nitrocellulose membranes (Thermo Scientific), blocked (5% skim milk powder; Sigma), and probed with anti-FLAG (Sigma), anti-CsrS (6 (link), 12 (link)), or anti-His (Abcam) antibodies. Proteins were visualized and analyzed using an Odyssey infrared imaging system (LI-COR, Lincoln, NE) and Image Studio software monitoring incubation with fluorescent secondary antibodies (Thermo Scientific).
Nupage tris acetate gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE Tris-Acetate gels are precast polyacrylamide gels used for protein electrophoresis. They are designed for the separation and analysis of proteins under denaturing conditions.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (8 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Immobilon fl membrane, by Merck Group (3 mentions)
Donkey anti goat igg irdye680rd, by LI COR (3 mentions)
Donkey anti mouse igg irdye800cw secondary antibodies, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Merck Group (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Image studio lite software, by LI COR (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Imagequant 5, by GE Healthcare (2 mentions)
Anti fancd2 antibody, by Novus Biologicals (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
58 protocols using nupage tris acetate gel
1
SDS-PAGE and Western Blot Analysis
2
Western Blotting Protocol for Protein Analysis
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Western blotting was performed according to standard protocols using IRDye (LI-COR) secondary antibodies. 3–8% NuPAGE Tris-Acetate gels (Thermo Fischer Scientific) were used to separate ELYS by SDS-PAGE. For other proteins, 4–12% NuPAGE Bis-Tris gels were used. After transfer to PVDF or nitrocellulose, the membranes were incubated in blocking buffer (3% BSA in TBS-T (24.8 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% (v/v) Tween 20)) for 1 h at room temperature and with primary antibodies (Appendix Table S2 ) overnight at 4 °C. Incubation with IRDye-labeld secondary antibodies (Appendix Table S2 ; diluted 1:10,000 in blocking buffer) for 1 h at room temperature was followed by three washing steps with TBS-T. Proteins were detected using LI-COR Odyssey-CLx imaging system and analyzed by Image Studio Lite software (LI-COR). For statistical analyses, ordinary one-way ANOVA, followed by Brown-Forsythe and Welch tests were performed using GraphPad Prism 8 software and a confidence interval of 95% was set.
3
Western Blot Analysis of Protein Targets
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Lysates (25 μg) were separated on 3%–8% NuPage Tris-acetate gels (Thermo Fisher, Carlsbad, CA, USA) and transferred to polyvinylidene fluoride (PVDF) (Thermo Fisher, Carlsbad, CA, USA). Membranes were probed with UNC596 antibody (1:2,000, provided by John Riordan and CFFT) and α-tubulin (1:10,000, Cell Signaling Technologies, Danvers, MA, USA) or α-Na/K ATPase (1:100,000, Abcam, Cambridge, MA, USA) followed by α-rabbit IRDye680 (1:15,000) and α-mouse IRDye800 (1:15,000) and imaged on a LI-COR Odyssey.
4
Western Blot Analysis of Keratinocytes
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Feeder layer was removed in 20 mM cold PBS/EDTA. Keratinocytes were collected by scraping in 1× RIPA buffer (Sigma Aldrich) supplemented with phosphatase and Protease Inhibitor Cocktail (Thermo Fisher). BCA kits (Pierce) were used to quantify the total protein amount. The same number of proteins was loaded in 4–12% NuPAGE Bis-Tris Gels or 10% NuPage Tris-Acetate Gels (Thermo Fisher) and transferred 100 V at 4 °C for 2 h onto nitrocellulose membrane (Millipore). Membranes were treated with blocking solution (5% (w/v) non-fat milk in 0.01% (v/v) Tween-20 in PBS 1×). Primary antibodies were diluted in blocking solution as indicated in Supplementary Table 1 and added overnight at 4 °C to the membranes. Secondary antibodies were diluted in Blocking solution as indicated in Supplementary Table 1 and added to the corresponding membranes for 1 h at room temperature. Signal was visualized with Clarity Western ECL substrate (Bio-Rad) using ChemiDoc (Bio-Rad) and ImagLabs software. Gray background on the images was homogeneously added for graphical purpose. Uncropped blots can be found in the Source data file.
5
Quantitative Cardiac Protein Analysis
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Five different biological replicates (tissue samples) were generated from the specific regions of the heart. Tissue chunks were harvested, snap frozen, and kept at -80 °C until lysis. Tissue pieces (50 mg) were aliquoted in 2-mL reinforced tubes containing 2.8-mm ceramic beads and lysed with Fisherbrand Bead Mill 24 homogenizer using a lysis solution containing radioimmunoprecipitation assay lysis buffer (9806; Cell Signaling Technology), protease inhibitors (P8340; Sigma-Aldrich), and phosphatase inhibitors (PhosSTOP; Sigma-Aldrich). All the lysates were clarified twice with ultracentrifugation, aliquoted, and kept at -20 °C. Rapid Gold bicinchoninic acid assay kit (Thermo Fisher Scientific) was used to quantify protein concentration. Proteins were denatured at 70 °C for 10 minutes in SDS, and 10 μg of samples were loaded on either 4% to 12% NuPAGE Bis-Tris gels or 3% to 8% NuPAGE Tris-Acetate gels (Thermo Fisher Scientific). After gel electrophoresis, proteins were transferred to 0.45-μM polyvinylidene difluoride membranes using a wet-transfer system (XCell II Blot Module). Membranes were subsequently blocked with 5% milk for 1 hour at room temperature and incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used: extracellular proteins: collagen 1 (ab138492; Abcam), collagen 3B).
6
Purification and Analysis of RAD51AP1 Complex
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The peGFP-RAD51AP1 expression vector is based on peGFP-C1 (Clontech) and has been described previously (Modesti et al. 2007) . RAD54/RAD51AP1 double KO cells were transfected with peGFP-C1 or peGFP-RAD51AP1 and Lipofectamine2000 (Invitrogen). Twenty-four hours after transfection, cells were subjected to a medium change or treated with 0.5 µM MMC for 2 hours. Cells were washed twice with warm PBS, fresh medium was added, and cells were incubated for the times indicated. Cells were lysed in chilled lysis buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 0.5% NP-40, supplemented with EDTA-free protease inhibitor cocktail (Roche) and HALT phosphatase inhibitors (Thermo Fisher Scientific). For 1.5x10 6 cells, 25 μl of GFP-Trap ® dynabeads (ChromoTek) were used to trap the ectopic proteins. Protein lysates were diluted to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, and 0.1unit DNase I (Gold Biotechnology) per µg protein, and mixed with the equilibrated beads at 4°C for 1 h with gentle rotation. The GFP-Trap ® dynabeads were washed three times with 500 µl binding buffer, bound protein complexes were eluted in 40 µl 2× LDS buffer (Thermo Fisher Scientific) and fractionated on 7% NuPAGE Tris-Acetate gels (Thermo Fisher Scientific) and for Western blot analysis.
7
Western Blot Analysis Protocol
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Cell lysates were prepared as described, added to 5X Laemmli buffer and heated at 95ºC for 5 min. Samples were run on Tris-Glycine or NuPAGE Tris-Acetate gels (Invitrogen) and transferred to PVDF membranes at 35V for 2 hr. Membranes were blocked in PBS-0.5%Tween-5%milk for 1 hr and stained with primary antibodies (1:250-1:2000) for 1 hr or overnight, followed by staining with secondary antibodies (1:10,000) conjugated with HRP (Jackson) for 1 hr. After incubation with ECL (Pierce, Thermo Fisher Scientific), blots were exposed to film. ImageQuant 5.2 (Molecular Dynamics) was used for quantification.
8
Detecting Monoubiquitinated FANCD2 Protein
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To detect the mobility‐shifted monoubiquitinated FANCD2 band, lysate proteins were separated on 3–8% NuPAGE Tris‐Acetate gels (Invitrogen) and transferred to nitrocellulose membranes. FANCD2 was detected via immunoblotting with an anti‐FANCD2 antibody (Novus Biologicals, Littleton, CO, USA).
9
SDS-PAGE and Immunoblotting Optimization
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Standard 10% to 14% SDS-PAGE gels were used for separation of most proteins except for HTT, which was better analyzed by NuPAGE Tris-Acetate gels from Invitrogen specially formulated for detection of proteins with large molecular weight. The boiled samples were separated on SDS-PAGE and transferred to nitrocellulose membranes from Millipore. After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 for 1 hour, membranes were incubated with primary antibodies. Secondary antibodies conjugated with Alexa-800 or Alexa-680 (Invitrogen) or HRP (KPL) were used and the signals were detected by the Odyssey Infrared Imaging System and quantified by Odyssey Application Software 3.0 or by densitometry of the digital images using ImageJ software (NIH).
10
Western Blot Analysis Methodology
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Cell lysates were prepared as described, added to 5× Laemmli buffer, and heated at 95°C for 5 min. Samples were run on Tris-glycine or NuPAGE Tris-acetate gels (Invitrogen) and transferred to polyvinylidene difluoride membranes at 35 V for 2 h. Membranes were blocked in PBS–0.5% Tween–5% milk for 1 h and stained with primary antibodies (1:250–1:2,000) for 1 h or overnight, followed by staining with secondary antibodies (1:10,000) conjugated with HRP (Jackson ImmunoResearch) for 1 h. After incubation with ECL substrate (Pierce/Thermo Fisher Scientific), blots were exposed to film. ImageQuant 5.2 (Molecular Dynamics) was used for quantification. The following primary antibodies were used for immunostaining: mouse anti–β-actin (A2228; Sigma-Aldrich), mouse anti-MYC (9E10, sc-40; Santa Cruz Biotechnology), rabbit anti–histone H3 (9715; Cell Signaling Technology), rabbit anti–histone H3K4me1 (ab8895; Abcam), rabbit anti–histone H3K27Ac (ab4729; Abcam), anti–phospho-Chk2 (2661, rabbit; Cell Signaling Technology), rabbit anti–phospho-RPA32 (S4/S8, A300-245A; Bethyl Laboratories), rabbit anti–phospho-CHK1 (2341S; Cell Signaling Technology), rabbit anti-UVSSA (GTX106751; GeneTex), rabbit anti-UVSSA (NBP1-32598; Novus Biologicals), and mouse S9.6 (ENH001; KeraFAST). HRP-conjugated secondary goat antirabbit and goat antimouse antibodies were obtained from Jackson ImmunoResearch.
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