Ab124974

Manufactured by Abcam

Sourced in United Kingdom

Ab124974 is a monoclonal antibody targeting the human GAPDH protein. It is suitable for use in various immunoassay applications.

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Lab products found in correlation

5 protocols using ab124974

Colonic Epithelial Cell Apoptosis Pathways

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Primary colonic epithelial cell and NCM460 cell homogenates were created using RIPA buffer. Protein concentration was quantified by the BCA kit (P0010, Beyotime Biotechnology, Shanghai, China). An equal amount of protein (40 mg) was separated by 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Then the proteins were probed using the primary antibodies, Bax (1:1000, 50,599–2-Ig; Proteintech, Rosemont, USA), active and pro Caspase-3 (1:1000, A19654; ABclone, Wuhan, China), Caspase 8 (1:1000, 13,423–1-AP; Proteintech), iNOS (1:500, ab178945; Abcam, Cambridge, UK), NOX1 (1:500, ab131088), and HDAC2 (1:1000, ab124974). After the corresponding secondary antibodies were incubated, the bands were detected with ultra-sensitive ECL reagents (P0018FS; Beyotime Biotechnology, Shanghai, China).

Chen L., Chu H., Hu L., Li Z., Yang L, & Hou X. (2022). The role of NADPH oxidase 1 in alcohol-induced oxidative stress injury of intestinal epithelial cells. Cell Biology and Toxicology, 39(5), 2345-2364.

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Chromatin Immunoprecipitation Assay

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5 μl of protein A Dynabeads (10001D, Invitrogen) were used in each MOWChIP assay. The beads were washed twice with IP buffer (20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100) and resuspended in 150 μl of IP buffer. 1 μg of Pol II or TF antibody was added into the bead suspension for each assay using 10⁵ cells/nuclei, while 0.5 μg of antibody was added for each assay using 5 × 10⁴ or fewer cells/nuclei. We used the following antibodies in this work: anti-Pol II-total (ab817, lot GR3271868-2, Abcam), anti-Pol II-S5 (ab5131, lot GR3202335-5, Abcam), anti-Pol II-S5 4H8 (ab5408, lot GR3325973-4, Abcam), anti-EGR1 (sc-101033, lot A1618, Santa Cruz), anti-MEF2C (sc-365862, lot B0818, Santa Cruz), anti-HDAC2 (ab124974, lot GR97402-7, Abcam). The suspension was incubated on a rotator at 4°C for 2 h. The beads were then washed with IP buffer twice and resuspended in 5 μl of IP buffer for loading into the device chamber.

Liu Z., Naler L.B., Zhu Y., Deng C., Zhang Q., Zhu B., Zhou Z., Sarma M., Murray A., Xie H, & Lu C. (2022). nMOWChIP-seq: low-input genome-wide mapping of non-histone targets. NAR Genomics and Bioinformatics, 4(2), lqac030.

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Western Blot Analysis of HDAC Proteins

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Proteins were separated on Criterion Stain-Free 4–20% gels (Biorad 567-8095) at 200V for 50 min. Proteins were transferred to Low Fluorescence PVDF membrane (Biorad 162-0264) at 0.14 amps for 60 min. Gels and membranes were imaged with a Chemidoc XRS system (Biorad 170-8265) for quality control purposes. Membranes were processed as follows: blocked in Tris buffered saline + Tween 20 (TBST, 0.1% Tween 20) containing 5% milk (Biorad 170-6404) at room temperature for 1 hr (note-rest of the protocol performed at room temperature), washed in TBST, incubated with primary antibodies in TBST containing 1% milk (HDAC1: Thermo Fisher PA1-860 1:5000, HDAC2: Abcam ab124974 1:5000, HDAC3: Abcam ab32369 1:5000, HDAC6: Santa Cruz sc11420 1:5000, HDAC8: Abcam ab187139 1:5000, GAPDH: Abcam ab8245 1:50000, acetyl histone H3 lysine 9: EMD Millipore 06-942-S 1:4000, acetyl histone H4 lysine 12: EMD Millipore 07-595 1:4000) for 1 hr, washed in TBST, incubated with secondary antibody in TBST containing 1% milk (anti-rabbit-HRP: Cell Signaling #7074S 1:5000, anti-mouse-HRP: Cell Signaling #7076S 1:5000) for 1 hr, washed in TBST, developed with ECL prime western blotting detection reagent (GE RPN2232), and visualized with a Chemidoc XRS system.

Wey H.Y., Gilbert T.M., Zürcher N.R., She A., Bhanot A., Taillon B.D., Schroeder F.A., Wang C., Haggarty S.J, & Hooker J.M. (2016). Insights into neuroepigenetics through human histone deacetylase PET imaging. Science translational medicine, 8(351), 351ra106.

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Histone Acetylation Dynamics Analysis

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Cells were stimulated for 30 min or 5 h with butyrate, propionate, or TSA, washed and lysed directly in Laemmli sample buffer (62 mM Tris/HCl pH 6.8; 2% SDS; 10% glycerol; 5% 2-mercaptoethanol; 0.01% bromophenol blue). Cell lysates were briefly sonicated, separated on 15% SDS-polyacrylamide gels and transferred to Immobilon-P polyvinylidene difluoride (PVDF) transfer membranes (0.45 μm, Millipore, Amsterdam, The Netherlands). Blots were blocked with PBS/2% milk/0.1% Tween20 and immunostained o/n at 4^°C with a polyclonal rabbit antibody against acetylated-lysines (#9441, Cell Signaling Technology, Bioké, Leiden, The Netherlands) at a 1:1,000 dilution. Other polyclonal antibodies used were directed against acetyl-H3K9 (Merck, 07-352), acetyl-H4K5 (Merck, 07-327), acetyl-H4K16 (Merck, 07-329), HDAC1 (Abcam, ab109411), HDAC2 (Abcam, ab124974) and HDAC3 (Abcam, ab16047). As a secondary antibody, HRP-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was used. As a loading control, GAPDH antibody (Sigma-Aldrich) was used at 1:10,000 dilution. Signal was visualized with the SuperSignal West Pico Chemiluminescent system (Thermo Scientific, Rockford, IL, USA) in a ChemiDoc MP (Biorad).

Lin M.Y., de Zoete M.R., van Putten J.P, & Strijbis K. (2015). Redirection of Epithelial Immune Responses by Short-Chain Fatty Acids through Inhibition of Histone Deacetylases. Frontiers in Immunology, 6, 554.

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ChIP Assay for Histone Modifications

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ChIP experiments were performed by the SimpleChIP® Enzymatic Chromatin IP Kit (product #9002; CST, Massachusetts, USA) as previously described (Lu et al. 2013 (link)). Chromatin samples that cross-linked with proteins were put to react with anti-HADC2 (ab124974, Abcam), anti-RNA polymerase II (ab238146, Abcam), anti-histone H3 (acetyl K9) (ab32129, Abcam), and anti-histone H3 (acetyl K27) (ab177178, Abcam). The precipitated genomic DNA was amplified using the primers of the NOX1 promotor region listed in Supplementary Table 2.

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