For immunocytochemistry: mouse monoclonal anti-cardiac troponin T antibody (cTnT; Thermo Scientific, Rockford, IL, USA), goat polyclonal anti-OCT4 antibody (R&D systems, Minneapolis, MN, USA), rabbit polyclonal anti-Ki67 antibody (Abcam, Cambridge, UK). FITC, Cy3 or Cy5 conjugated secondary antibodies were used (Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with Hoechst33258 (TheremoFisher, Waltham, MA, USA). For Western blot: rabbit polyclonal anti-actin was obtained from Cell Signaling (Danvers, MA, USA), rabbit polyclonal anti-α-tubulin, rabbit polyclonal anti-ɣ-H2AX and rabbit polyclonal anti-pSer2 RNA Pol II antibodies were obtained from Abcam (Cambridge, UK). Mouse monoclonal anti-p53 and goat polyclonal anti-cleaved caspase-9 antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA) and rabbit polyclonal anti-MCL-1 antibody was obtained from Cell Signaling. Secondary peroxidase-labelled anti-mouse and anti-rabbit were obtained from (GE Healthcare, Marlborough, MA, USA). Secondary peroxidase labeled anti-goat was obtained from Santa Cruz. Dinaciclib (SCH 727965) was obtained from Adoq Bioscience (Irvine, CA, USA).
Rabbit polyclonal anti α tubulin
Manufactured by Abcam
Sourced in United States
Rabbit polyclonal anti-α-tubulin is an antibody product designed for the detection of α-tubulin, a major component of the cytoskeleton. It is raised in rabbits and recognizes the α-tubulin protein.
Automatically generated - may contain errors
Lab products found in correlation
Cy5 conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions)
Alexafluor 680 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ecltm prime western blotting detection reagent, by GE Healthcare (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Analyzer fusion solo 4tm system, by Vilber (1 mentions)
Goat anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
Rabbit monoclonal anti αsma, by Cell Signaling Technology (1 mentions)
Fusion solo s imaging system, by Vilber (1 mentions)
Bovine serum albumin bsa, by Fujifilm (1 mentions)
5 protocols using rabbit polyclonal anti α tubulin
1
Immunocytochemistry and Western Blot Protocols
2
Immunofluorescence Antibody Validation
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Rabbit polyclonal anti-EZH2, rabbit polyclonal anti-PCAF were purchased from Cell Signaling Technology (Beverly, MA, USA); goat polyclonal anti-BubR1, rabbit polyclonal anti-H3K27me3 and rabbit polyclonal anti-α-tubulin were purchased from Abcam (Cambridge, MA, USA); human CREST antibody was purchased from Fitzgerald; and rabbit polyclonal anti-Myc was purchased from MBL (Nagoya, Japan). Alexa Fluor 488 donkey anti-rabbit IgG (H + L), Alexa Fluor 568 donkey anti-goat IgG (H + L) were purchased from Invitrogen (Eugene, OR, USA); and CY5-conjugated goat anti-human IgG was purchased from Jackson ImmunoResearch.
3
Western Blot Analysis of FLAG-Tagged Proteins
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Cells were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the DC protein assay (Bio-Rad) and proteins ran on 5–12% acrylamide gradient gels. For FLAG epitope detection, the transferred membrane was incubated with mouse anti-FLAG M2 monoclonal antibody (Stratagene, UK) at 1:1000 dilution, using IRDye® 800CW goat anti-mouse (Li-cor GmbH, Germany) at 1:2000 dilution as secondary antibody. For loading controls, rabbit polyclonal anti α-tubulin (Abcam, UK) at 1:2000 dilution was used as primary antibody, and then Alexa Fluor® 680 Goat anti-rabbit (Invitrogen, USA) at 1:5000 dilution as secondary antibody. Blots were scanned at 700 and 800 nm channels for α-tubulin and anti-FLAG, respectively, using an Odyssey imager (Li-cor Bioscience, Germany).
4
Western Blot Protein Analysis Protocol
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Whole-cell lysate samples were separated by SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore, Bedford, MA, USA) by electroblotting at 15 V for 60 min. For blocking, the membrane was incubated in Tris-buffered saline containing 0.05% Tween 20 and 3% BSA (TBST-3% BSA). Blots were probed with appropriate antibodies (Supplementary Table 1 ), and the signals were visualized by chemiluminescence. All antibodies were used at a 1:1000 (first antibodies—Rabbit polyclonal anti-EGFP; Cat# A11122; Life Technologies: Rabbit polyclonal anti-α-tubulin; Cat# ab15246; Abcam, Cambridge, MA, USA) or 1:2,000 (second antibody—Donkey anti-rabbit IgG-horseradish peroxidase (HRP)-conjugate; Cat# NA934V; GE Healthcare, Buckinghamshire, UK) dilution in TBST-0.1% BSA for 1 h at room temperature. After washing by TBST for 1 h at room temperature, HRP-dependent luminescence was developed with ECLTM Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging Analyzer Fusion Solo 4TM system (Vilber Lourmat, Eberhardzell, Germany). Uncropped scans of the blots were shown in Supplementary Fig. 3 .
5
Western Blot Analysis of Cellular Proteins
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Denatured cell lysates (30 µg of total proteins in Figs. 5A and S5B ; 20 µg of proteins in Figs. 3D , 4C , S3B and S4B ; or 10 µg of total protein in Fig. S1 ) were separated on 10.5% or 12% SDS-PAGE gel depending on the experiment, followed by transfer onto PVDF membranes (Merck KGaA). The membranes were then blocked with 3% bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation) for 30 min at room temperature and incubated with appropriate primary antibodies diluted in 3% BSA (Nacalai Tesque, Inc.): Rabbit monoclonal anti-αSMA (1:1,000; product no. 19245; Cell Signaling Technology, Inc.), rabbit polyclonal anti-TAGLN/Transgelin (SM22α; 1:2,000; product code ab14106; Abcam), rabbit polyclonal anti-human IgG-Fc fragment (1:5,000), and rabbit polyclonal anti-α-tubulin (1:10,000; product code ab4074; Abcam) overnight at 4°C. The membranes were then incubated with goat anti-rabbit IgG HRP-linked antibody (1:5,000; product no. 7074S; Cell Signaling Technology, Inc.) for 1 h at room temperature. The target proteins were detected using an Enhanced Chemiluminescence Kit (ECL detection reagent; Cytiva) and visualized with a Fusion Solo S Imaging System (SOLO.6S.EDGE; Vilber Lourmat).
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