Ab154179

Manufactured by Abcam

Sourced in China

Ab154179 is a product offered by Abcam. It is a laboratory equipment item, but a detailed description while maintaining an unbiased and factual approach is not available.

Automatically generated - may contain errors

Lab products found in correlation

Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Ab181090, by Abcam (1 mentions) Nitrocellulose membrane, by Merck Group (1 mentions) Ab178945, by Abcam (1 mentions) Ripa lysis buffer, by Merck Group (1 mentions) Pa5 99390, by Abcam (1 mentions) Mitotracker red, by Thermo Fisher Scientific (1 mentions) Ab192890, by Abcam (1 mentions) Ab109012, by Abcam (1 mentions) Superoxide dismutase activity assay kit, by Abcam (1 mentions) Tempol, by Merck Group (1 mentions) Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Mitotracker green, by Thermo Fisher Scientific (1 mentions) Mitosox red, by Thermo Fisher Scientific (1 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions) Mda assay kit, by Beyotime (1 mentions) Dcfh da, by Beyotime (1 mentions) Ros assay kit, by Beyotime (1 mentions)

3 protocols using ab154179

Protein Expression Analysis in Macrophages

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Protein was extracted from the tissue homogenate and macrophages using RIPA lysis buffer (Sigma-Aldrich). NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to prepare the cytosolic fraction and nuclear extracts. Equivalent quantities (25 µg) of protein were separated by SDS-PAGE gel, transferred onto a nitrocellulose membrane (Millipore), blocked with 5% skim milk overnight at 4 °C, followed by overnight incubation with primary antibodies against Atox1 (1:10000; Abcam, ab154179), p47phox (1:1000; Abcam, ab181090), NLRP3 (1:500; Abcam, ab263889), Caspase 1 p20 (1:1000; Invitrogen, PA5-99390), iNOS (1:20000; Abcam, ab178945), IL-12p40 (1:1000; Abcam, ab133752), IL-10 (1:2000; Abcam, ab1333575), Arg-1 (1:1000; Abcam, ab2333548), Lamin B1 (1:1000; Abcam, ab229025), and β-actin (1:5000; Proteintech Group, Inc., 60066-1-AP) at 4 °C. Subsequently, the membranes were washed thrice with Tris-buffered saline with 0.1% Tween-20 and incubated with the HRP-conjugated secondary antibody (1:10000; ZSGB-BIO, ZB-2301, ZB-2305) for 1 h at 37 °C. Signals were visualized with an enhanced chemiluminescence system.

Chen M., Chen Y., Fu R., Liu S., Li H, & Shen T. (2024). Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway. Journal of Translational Medicine, 22, 497.

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Oxidative Stress-Induced Apoptosis Assays

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Annexin V-FITC Apoptosis Detection Kit (Invitrogen, China), Ammonium tetrathiomolybdate (TTM) (Sigma-Aldrich, USA), TUNEL assay (Beyotime, China), ROS Assay kit (Beyotime, China), ROS Fluorescent Probe Kit (KeyGEN, China), MDA Assay Kit (Beyotime, China), GSH and GSSG Assay Kit (Beyotime, China), Tempol (Sigma-Aldrich, USA), Cell Counting Kit-8 (Dojindo, Japan), DCFH-DA (Beyotime, China), MitoSOX Red (Invitrogen, China), JC-1 (Invitrogen, China), Mito-Tracker Red, Mito-Tracker Green and Lysosome-Tracker Red (Invitrogen, China), Cell mitochondria isolation kit (Beyotime, China), Superoxide Dismutase Activity Assay kit (ab65354; Abcam).
Antibodies were from various sources, including GAPDH (5174; Cell Signaling Technology), Atox1 (22641-1-AP; Proteintech), Atox1 (ab154179; Abcam), NeuN (ab177487; Abcam), Cleaved Caspase-3 (9661; Cell Signaling Technology), Bax (ab32503; Abcam), Bcl-2 (ab182858; Abcam), DJ-1 (ab76008; Abcam), TOMM20 (ab283317, Abcam), TOMM20 (ab186735, Abcam), LC3-Ⅱ (ab192890, Abcam), SQSTM1/p62 (ab109012, Abcam), PINK1 (23274-1-AP, Proteintech), PRKN (14060-1-AP, Proteintech), COX4 (11242-1-AP, Proteintech), FLAG (ab205606; Abcam).

Zhao P., Shi W., Ye Y., Xu K., Hu J., Chao H., Tao Z., Xu L., Gu W., Zhang L., Wang T., Wang X, & Ji J. (2024). Atox1 protects hippocampal neurons after traumatic brain injury via DJ-1 mediated anti-oxidative stress and mitophagy. Redox Biology, 72, 103156.

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Quantification of Recombinant Protein HAH1

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Protein content was measured by Western Blot analysis. HAH1 was stained with a rabbit monoclonal anti-HAH1 antibody (Abcam: ab154179, diluted to 53.2 ng/mL). GAPDH was used as reference (rabbit polyclonal anti-GAPDH antibody, Abcam: ab9485, diluted at 1:2,000). Goat anti-rabbit IgG (whole molecule)-peroxidase secondary antibody (Sigma:A0545) was used, diluted at 1:80,000. For detection, LiteAblot EXTEND chemiluminescent substrate (EuroClone) was used. Purified recombinant HAH1 was used as standard for the quantification. HAH1 was expressed with a 6-His tag at C-terminal part in E. coli BL21(DE3) gold competent cells for 4 h at 37 °C. After cell lysis, a first purification step was performed using a HiTrap chelating (GE Healthcare) column charged with Zn(II). The protein was eluted in 20 mM Na2HPO4, 0.5 M NaCl, 5 mM imidazole, 20 mM EDTA, pH=8, buffer. After buffer exchange and digestion with Factor Xa protease (New England Biolabs) for 24 hr at 25 °C, the protein was separated from the affinity tag in the HiTrap column charged with Zn(II).

Luchinat E., Secci E., Cencetti F, & Bruni P. (2016). Sequential protein expression and selective labeling for in-cell NMR in human cells. Biochimica et biophysica acta, 1860(3).

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