Recombinant mouse IFN-γ (485 MI/CF) was obtained from R&D systems. 1400W, and Dimethyloxalylglycine (DMOG) were obtained from Cayman Chemical. Pam3CysK4 (PAM) was obtained from EMC Microcollections. Ascorbate, citrulline, and S-Nitroso-N-acetylpenicillamine (SNAP) were obtained from Sigma-Aldrich. The following primary antibodies were used: HIF-1α (NB100-479, Novus Biologicals), IL-1b (AF-401-NA, R&D systems), and the following Cell Signaling Technology antibodies: HIF-1α (D2U3T), RelA (D14E12), RelB (C1E4), NF-kB1 (D4P4D), IkBa (L35A5), α/β-Tubulin (2148), Histone H3 (D1H2), and β-actin (13E5).
Hif 1α nb100 479
Manufactured by Novus Biologicals
Sourced in United States
HIF-1α (NB100-479) is a product offered by Novus Biologicals for laboratory research purposes. It is an antibody that specifically targets the hypoxia-inducible factor-1 alpha (HIF-1α) protein. HIF-1α is a subunit of the HIF-1 transcription factor, which plays a crucial role in the cellular response to low oxygen levels.
Automatically generated - may contain errors
Lab products found in correlation
Citrulline, by Merck Group (1 mentions)
Ascorbate, by Merck Group (1 mentions)
Recombinant mouse ifn γ 485 mi cf, by R&D Systems (1 mentions)
Dimethyloxalylglycine dmog, by Cayman Chemical (1 mentions)
Il 1b af 401 na, by R&D Systems (1 mentions)
β actin 13e5, by Cell Signaling Technology (1 mentions)
S nitroso n acetylpenicillamine snap, by Merck Group (1 mentions)
Ab14715, by Abcam (1 mentions)
Ab199, by Abcam (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Hif1α 610958, by BD (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Las 3000 imager, by Fujifilm (1 mentions)
Ab107257, by Abcam (1 mentions)
Ab40800, by Abcam (1 mentions)
Hif 2α nb100 122, by Novus Biologicals (1 mentions)
Hif 1α gtx127309, by GeneTex (1 mentions)
Ab1791, by Abcam (1 mentions)
4 protocols using hif 1α nb100 479
1
Mouse Interferon-gamma Signaling Assay
2
Western Blot Analysis of HIF-1α and HIF-2α
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Whole cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer supplemented with a complete protease inhibitor cocktail (Roche Life Science, Penzberg, Germany). Equal amounts of sample (80-100 μg protein) were diluted in Laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA) with dithiothreitol (DTT) and were boiled for 5 min. Lysates were separated on 7.5% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad Laboratories) and proteins were transferred to polyvinylidene fluoride (PVDF) membranes using a Transblot Turbo System (Bio-Rad Laboratories). Membranes were blocked in 5% non-fat dry milk/Tris buffered saline with Tween 20 (TBS-T), and incubated overnight with the following primary antibodies: HIF-1α (NB100-479, 1:500 dilution, Novus Biologicals, Centennial, CO, USA), HIF-1α (610958, 1:500 dilution, BD Bioscience, San Jose, CA; USA), HIF-2α (ab199, 1:500 dilution, Abcam), Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA) (ab14715, 1:4000 dilution, Abcam). After washing, membranes were incubated for 1 h with secondary antibodies (1:5000 dilution, Jackson ImmunoResearch, West Grove, PA, USA). Images were acquired using a LAS-3000 Imager (Fujifilm, Tokyo, Japan). Band intensity was quantified using Fiji [38 (link)].
3
Antibody Characterization for Western Blot Analysis
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Antibodies used for western blot analysis are as follows: HIF-1α (GTX127309) from GeneTex; HIF-1α (NB100-479) and HIF-2α (NB100-122) from Novus Biologicals; HIF-2α (#7096), pVHL (#2738), FASN (#3180) and ubiquitin (#3936) from Cell Signaling; Elongin C (sc-135895), CUL2 (sc-166506), ARNT (sc-5580), SREBP1 (sc-366), and normal mouse and rabbit IgG (sc-2025 and sc-2027 respectively) from Santa Cruz Biotechnology; CAD (ab40800), RNA polymerase II large subunit CTD (ab5408), and CA9 (ab107257) from Abcam; β-actin (A5441) and anti-Flag M2 (F1804) from Sigma-Aldrich; p65 (610869) from Becton Dickinson (BD) and REDD1 (A500-001A) from Bethyl Laboratories. For immunofluorescence, antibodies against pVHL (BD, 556347) or FASN (#3180) were used. For immunoprecipitation, antibodies against pVHL (BD, 556347), FASN (Santa Cruz Biotechnology, sc-55580) or CAD (Abcam, ab40800) were used.
4
Quantifying Hypoxia-Induced HIF-1α and Targets
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To measure HIF-1α, cells were exposed to oxygen conditions for 6 h, as HIF-1α protein levels peak after approximately 6 h of hypoxia exposure and then gradually decline [39 (link),40 (link)]. For downstream HIF-1 target genes, cell lysates were generated following 18 h of normoxia, chronic hypoxia and intermittent hypoxia (near the peak of HIF-1 target gene expression [39 (link)]). Lysates were prepared using RIPA buffer containing protease inhibitors (Roche complete, Sigma-Aldrich, Castle Hill, NSW Australia) and resolved using SDS-PAGE and western blotting as before [41 (link)]. Antibodies were as follows: HIF-1α (NB100-479, Novus), Glut1 (ab115730, Abcam, Cambridge, MA, USA), hexokinase 2 (TA325030, Origene), Histone H3 (ab1791, Abcam), PHD2 (4835, Cell Signaling Technologies, Danvers, MA, USA) and LDHA (3582T, Cell Signaling Technologies).
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