Yeast strains of the indicated genotypes (and their wild-type counterparts) were grown in YPD either at permissive or restrictive temperatures. Overnight-saturated cultures were diluted to an OD600 of 0.2 and allowed to grow until they reached an OD600 of 1. Five OD600 equivalents of cells were lysed using a modified TCA extraction method as described (Keogh et al., 2006a (link), 2006b (link)). 10–20 mg of the lysates were separated by SDS-PAGE and variously probed with the following antibodies: anti-FLAG-M2 [for FLAG tagged Spt6] (Sigma-Aldrich, F1804; 1:5000), anti-G6PDH (Sigma-Aldrich, A9521; 1:100,000), anti-histone H3K4me3 (EpiCypher, 13–0004; 1:5000), anti-histone H3K79me3 (Abcam, ab2651, 1:2500) anti-histone H3K36me3 (Abcam, ab9050, ab9050; 1:1000), anti-histone H3 (EpiCypher, 13–0001; 1:50,000), anti-Spt16 (gift from Tim Formosa University of Utah, 1:5000), anti-H3K36me2 (Active Motif, 39255; 1:1000), anti-Set2 (Generated in the Strahl lab, 1:5000), anti-RNAPII-Ser2P (Active Motif, Clone #3E10, 61084; 1:100), anti-H2BK123ub1 (Cell Signaling Technology, 5546; 1:2000), and anti-H2B (Active Motif, 39237; 1:2000). HRP-conjugated anti-rabbit (GE Healthcare, NA934V; 1:10,000) and anti-mouse secondary (GE Healthcare, NA931V; 1:10,000), antibodies were used at 1:1000 and proteins were detected using ECL Prime or enhanced chemiluminescence ECL (Amersham Biosciences).
Anti mouse secondary
Manufactured by GE Healthcare
Sourced in United States
The Anti-mouse secondary is a laboratory reagent used in various immunochemical techniques, such as Western blotting, ELISA, and immunohistochemistry. It is designed to specifically bind to primary antibodies raised against mouse antigens, enabling the detection and visualization of target proteins or cellular components. The Anti-mouse secondary is a critical tool for researchers studying mouse-derived biological samples or utilizing mouse monoclonal antibodies in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab2651, by Abcam (2 mentions)
Ab9050, by EpiCypher (2 mentions)
Anti h2b, by Active Motif (2 mentions)
Na934v, by GE Healthcare (2 mentions)
Anti g6pdh, by Merck Group (2 mentions)
Anti h3k36me2, by Active Motif (2 mentions)
Anti histone h3, by EpiCypher (2 mentions)
Anti flag m2 for flag tagged spt6, by Merck Group (2 mentions)
Anti histone h3k4me3, by EpiCypher (2 mentions)
Anti histone h3k79me3, by Abcam (2 mentions)
Anti histone h3k36me3, by Abcam (2 mentions)
Anti set2, by Active Motif (2 mentions)
Anti rnapii ser2p, by Active Motif (2 mentions)
Anti h2bk123ub1, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti rabbit, by GE Healthcare (2 mentions)
Ecl prime, by Cytiva (2 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (2 mentions)
Hybond p, by GE Healthcare (1 mentions)
Anti rad53 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti goat secondary antibody, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti mouse secondary
1
Yeast Protein Extraction and Immunoblotting
2
Yeast Protein Extraction and Immunoblotting
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Yeast strains of the indicated genotypes (and their wild-type counterparts) were grown in YPD either at permissive or restrictive temperatures. Overnight-saturated cultures were diluted to an OD600 of 0.2 and allowed to grow until they reached an OD600 of 1. Five OD600 equivalents of cells were lysed using a modified TCA extraction method as described (Keogh et al., 2006a (link), 2006b (link)). 10–20 mg of the lysates were separated by SDS-PAGE and variously probed with the following antibodies: anti-FLAG-M2 [for FLAG tagged Spt6] (Sigma-Aldrich, F1804; 1:5000), anti-G6PDH (Sigma-Aldrich, A9521; 1:100,000), anti-histone H3K4me3 (EpiCypher, 13–0004; 1:5000), anti-histone H3K79me3 (Abcam, ab2651, 1:2500) anti-histone H3K36me3 (Abcam, ab9050, ab9050; 1:1000), anti-histone H3 (EpiCypher, 13–0001; 1:50,000), anti-Spt16 (gift from Tim Formosa University of Utah, 1:5000), anti-H3K36me2 (Active Motif, 39255; 1:1000), anti-Set2 (Generated in the Strahl lab, 1:5000), anti-RNAPII-Ser2P (Active Motif, Clone #3E10, 61084; 1:100), anti-H2BK123ub1 (Cell Signaling Technology, 5546; 1:2000), and anti-H2B (Active Motif, 39237; 1:2000). HRP-conjugated anti-rabbit (GE Healthcare, NA934V; 1:10,000) and anti-mouse secondary (GE Healthcare, NA931V; 1:10,000), antibodies were used at 1:1000 and proteins were detected using ECL Prime or enhanced chemiluminescence ECL (Amersham Biosciences).
3
Phosphorylation Analysis of Yeast Proteins
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Samples were prepared by trichloroacetic acid (TCA) extraction. Cells were collected by centrifugation and fixed with 20% TCA. Cell breakage was performed with glass beads in a FastPrep machine (MPBio) (three 20‐s cycles at power setting 6.5). Protein precipitation was attained by centrifugation at 2,500 g for 5 min at 4°C. After precipitation, the pellet was solubilized in 1 M Tris–HCl pH8 and SDS‐PAGE loading buffer and boiled for 10 min. Insoluble material was removed by centrifugation, and the supernatant was loaded in a 6% acrylamide gel. When separating Spc110 phospho‐bands, gels were supplemented with 30 μM of Phos‐Tag and 60 μM of MnCl2. For Net1 phosphorylation analysis, 10 μM of Phos‐Tag and 20 μM of MnCl2 were used. Proteins were transferred into a PVDF membrane (Hybond‐P; GE Healthcare) and blocked with 5% milk in PBS–Tween (0.1%). Anti‐Rad53 antibody was used at a 1:1,000 dilution (Santa Cruz), and the secondary anti‐goat antibody was used at a concentration of 1:5,000 (Santa Cruz). Anti‐HA antibody was used at a 1:2,500 dilution (Roche), and the secondary anti‐mouse was used at a concentration of 1:25,000 (GE Healthcare). After several washes in PBS–Tween, membranes were incubated with SuperSignal® West Femto (Thermo Scientific), followed by exposure to ECL Hyperfilm (GE Healthcare).
4
Western Blot Analysis of Caspase Activation
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The 10630 (1:10,000) and GN60622 (1:10,000) neoepitope antibodies against the p20 subunit of active Casp616 (link) and Tub∆Casp623 (link), respectively, were generated in our laboratory. The β-actin clone AC-15 (1:5000, Sigma-Aldrich), Casp6 p10 clone B93-4 (1:250, BD Canada, Mississauga, ON, CA), Casp3, and full-length α-tubulin (1:1000, Cell Signaling Technology Inc., Danvers, MA, USA) antibodies were diluted in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (Sigma-Aldrich). Secondary anti-mouse (1:5000, GE Healthcare Life Sciences, Baie D’Urfe, QC, CA) and anti-rabbit antibodies (1:5000, Dako, Burlington, ON, CA) conjugated to horseradish peroxidase (HRP) were used to detect immunoreactive proteins using ECL prime western blotting detection reagent (GE Healthcare Life Sciences) and Kodak BioMax MR film (Kodak, Rochester, NY, USA). Secondary anti-mouse conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories Inc., West Grove, PA) was developed with nitro-blue tetrazolium (ThermoSci) and 5-bromo-4-chloro-3′-indolyphosphate (ThermoSci). The western blots were scanned with an HP scanner and densitometry was performed using ImageJ software (NIH, Bethesda, MD) by rendering tiff images into an 8-bit format and measuring the intensity values above background. Images were not modified except to adjust the contrast for the entire blot.
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