Nb100 122

Manufactured by Novus Biologicals

Sourced in United States, United Kingdom, Germany

The NB100-122 is a laboratory equipment product offered by Novus Biologicals. It is a device designed for general laboratory use. The core function of this product is to provide a controlled environment for various scientific procedures and experiments.

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Lab products found in correlation

Nb100 479, by Novus Biologicals (8 mentions) Nb100 105, by Novus Biologicals (6 mentions) β actin, by Merck Group (5 mentions) Nb100 134, by Novus Biologicals (3 mentions) Anti hif 2α, by Novus Biologicals (3 mentions) Hif 2α, by Novus Biologicals (3 mentions) Nb100 110, by Novus Biologicals (3 mentions) Ripa lysis buffer, by Merck Group (3 mentions) Ab28455, by Abcam (2 mentions) Ab83690, by Abcam (2 mentions) Ab37583, by Abcam (2 mentions) Rabbit 2729, by Cell Signaling Technology (2 mentions) Mouse 5415, by Cell Signaling Technology (2 mentions) Sab1405798, by Merck Group (2 mentions) Hpa005857, by Merck Group (2 mentions) Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Horseradish peroxidase hrp conjugated antibodies, by Promega (2 mentions) Pierce ecl western blotting system, by Thermo Fisher Scientific (2 mentions) T4026, by Merck Group (2 mentions) Sc 2004, by Santa Cruz Biotechnology (2 mentions)

Close spelling variants of this product

Rabbit anti‐HIF‐2α (NB100‐122) (2 citations) HIF2α antibody (NB100-122) (3 citations) HIF-2α (NB100-122, (8 citations) Anti-HIF-2α (NB100-122) (7 citations)

60 protocols using nb100 122

Immunoprecipitation and Immunoblotting Assay for HIF-1α, HIF-2α, and p300 Proteins

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Whole cell lysates were prepared in modified RIPA buffer [50 mM Tris-HCl (pH 7.5), 1 mM β-mercaptoethanol, 150 mM NaCl, 1% Igepal, and protease inhibitor cocktail] and incubated overnight with the following antibodies (catalog number and supplier): HIF-1α (sc-10790, Santa Cruz); V5 epitope (NB600-381, Novus Biologicals), HIF-2α (NB100-122, Novus Biologicals), or p300 (NB500-161, Novus Biologicals) in the presence of protein A Sepharose beads (NBP1-97240, Novus Biologicals). After washing three times, the bound proteins were fractionated by SDS-PAGE, followed by immunoblot assays using antibodies against the following: HIF-1α (610958, BD Bioscience); HIF-2α (NB100-122, Novus Biologicals); and V5 epitope (R960-25, Invitrogen). Other antibodies used in immunoblot assays recognized PRDX2 (H00007001-M01, Novus Biologicals) and PRDX4 (NBP2-19778, Novus Biologicals), and GST (sc-459, Santa Cruz) and actin (sc-1616, Santa Cruz).

Luo W., Chen I., Chen Y., Alkam D., Wang Y, & Semenza G.L. (2016). PRDX2 and PRDX4 are negative regulators of hypoxia-inducible factors under conditions of prolonged hypoxia. Oncotarget, 7(6), 6379-6397.

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Immunocytochemical Analysis of Cellular Markers

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The procedures of immunocytochemistry were described previously^{19 (link)}. Nucleuses were stained with DAPI (1 μg/ml) or PI (2 μg/ml) containing RNase A (50 μg/ml). Samples were examined with a BZ-9000 inverted fluorescence microscope (Keyence). Anti-GFAP rabbit polyclonal antibodies (G4546, Sigma-Aldrich), anti-MBP rat monoclonal antibody (MAB386, Millipore), anti-Ki-67 rabbit polyclonal antibodies (AB9260, Millipore), anti-Hif1α rabbit polyclonal antibodies (NB100-479, Novus Biologicals), anti-Hif2α rabbit polyclonal antibodies (NB100-122, Novus Biologicals), Anti-CDKN2B (p15/INK4b) rabbit polyclonal antibodies (bs-4269R, Bioss Antibody), anti-RUNX1/AML1 rabbit polyclonal antibodies (ab23980, Abcam).

Tokumoto Y., Tamaki S., Kabe Y., Takubo K, & Suematsu M. (2017). Quiescence of adult oligodendrocyte precursor cells requires thyroid hormone and hypoxia to activate Runx1. Scientific Reports, 7, 1019.

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Antibody Detection of Metabolic Proteins

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Antibodies detecting HDAC1 (5356), HSP90 (4877), GST (2625), HA (3724), and normal IgG isotopic controls (rabbit-2729, mouse-5415) were purchased from Cell Signaling Technology. Antibodies detecting HIF1α (NB100-134) and HIF2α (NB100-122) were obtained from Novus, and antibodies against PCK1 (ab28455), G6PC (ab83690), and PFKL (ab37583) were from Abcam. D-fructose-1, 6-bisphosphate trisodium salt (47810), 2-hydroxyethyl agarose (A4018), and antibodies detecting FBP1 (SAB1405798 and HPA005857) were purchased from Sigma.

Li B., Qiu B., Lee D.S., Walton Z.E., Ochocki J.D., Mathew L.K., Mancuso A., Gade T.P., Keith B., Nissim I, & Simon M.C. (2014). Fructose-1, 6-bisphosphatase opposes renal carcinoma progression. Nature, 513(7517), 251-255.

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Protein Immunodetection in Hypoxic Cells

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Protein immunodetection was performed as previously ///described.49 Briefly, cells were lysed in 1.5X SDS buffer, and if exposed to hypoxia, they were lysed while inside the hypoxic chamber. Proteins (40 μg) were separated on 7.5% SDS‐polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Blots were blocked in 5% milk in Tris‐HCl/NaCl buffer and incubated with antibodies at 4°C overnight. Immunoreactive signals were revealed with horseradish peroxidase (HRP)‐conjugated antibodies (Promega) using the Pierce^TM ECL Western blotting system (Thermo Fisher Scientific). The antibody against HIF‐1α was produced in our laboratory and used at 1:1000. The antibody against HIF‐2α (NB100‐122) was purchased from Novus Biologicals (Littleton) and used at 1:1000. The antibody against OPN (AF 14‐33OP) was purchased from R&D System and used at 1:500. The antibody against β‐tubulin (T4026; Sigma‐Aldrich) was used at a 1:2000 dilution as a loading control.

Sadaghianloo N., Contenti J., Dufies M., Parola J., Rouleau M., Lee S., Peyron J., Fabbri L., Hassen‐Khodja R., Pouysségur J., Bost F., Jean‐Baptiste E., Dardik A, & Mazure N.M. (2020). Co‐culture of human fibroblasts, smooth muscle and endothelial cells promotes osteopontin induction in hypoxia. Journal of Cellular and Molecular Medicine, 24(5), 2931-2941.

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Immunohistochemical Profiling of Hypoxia

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Paraffin embedded tissue sections were deparaffinized and stained according to previously published protocols (27 (link)). Primary antibodies HIF-1 (anti-rabbit A300-286A; 1:100; Bethyl Laboratories), HIF-2 (anti-rabbit NB100-122; 1:100; Novus Biologicals), LOX (anti-rabbit ab174316; 1:500; Abcam) and PIMO (anti-rabbit; 1:100; Hypoxyprobe) were used.

Natarajan S., Foreman K.M., Soriano M.I., Rossen N.S., Shehade H., Fregoso D.R., Eggold J.T., Krishnan V., Dorigo O., Krieg A.J., Heilshorn S.C., Sinha S., Fuh K.C, & Rankin E.B. (2019). Collagen remodeling in the hypoxic tumor-mesothelial niche promotes ovarian cancer metastasis. Cancer research, 79(9), 2271-2284.

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HIF1α and HIF2α ChIP in VSMCs

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Hif1a^fl/fl or Hif1a^ΔSMC VSMCs treated with Ang II (1 μM) for 24 h were crosslinked in 1% formaldehyde in 1× PBS for 10 min. ChIP assays were performed for HIF1α (2 μg/IP, NB100-105; Novus Biologicals) or HIF2α (2 μg/IP, NB100-122; Novus Biologicals) using Simple ChIP Plus Kit (Cell Signaling Technology, Danvers, MA, USA) as previously described^{14 (link)}. The primers for ChIP assays are listed in Supplementary Table 1.

Qi D., Wei M., Jiao S., Song Y., Wang X., Xie G., Taranto J., Liu Y., Duan Y., Yu B., Li H., Shah Y.M., Xu Q., Du J., Gonzalez F.J, & Qu A. (2019). Hypoxia inducible factor 1α in vascular smooth muscle cells promotes angiotensin II-induced vascular remodeling via activation of CCL7-mediated macrophage recruitment. Cell Death & Disease, 10(8), 544.

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Immunohistochemical Evaluation of Cartilage Degradation

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Serial sections adjacent to those with representative
Safranin O-stained images were dewaxed in xylene and
rehydrated through a graded series of alcohols for
immunohistochemical evaluation. Briefly, after antigen
retrieval with boiling in sodium citrate buffer,
endogenous peroxidase activity was quenched in 3%
H₂O₂ for 15 min and washed
in phosphate buffered saline (PBS). Sections were then
blocked in serum for 30 min followed by incubation with
the primary antibody in humidified chamber at 4 °C
overnight. Biotinylated secondary antibody was added
for 30 min on the second day, followed by an
avidin-biotinylated horseradish peroxidase complex
according to the manufacturer’s directions
(Vectastain ABC Kit; Vector Laboratories, Burlingame,
CA, USA). Finally, peroxidase activity was revealed by
immersion in DAB substrate (Dako, Glostrup, Denmark).
The following primary antibodies were used: rabbit
anti-aggrecanase generated-aggrecan neoepitope NITEGE,
rabbit anti-MMP-generated aggrecan-neoepitope DIPEN
(gifts from Dr John S Mort), rabbit anti-MMP13
(ab75606; Abcam, Cambridge, MA, USA), rabbit
anti-ADAMTS5 (ab41037; Abcam), rabbit anti-HIF2α
(NB100–122; Novus Biologicals, Littleton, CO,
USA). TUNEL staining was carried out on paraffin
sections using ApopTag Plus Peroxidase in Situ
Apoptosis Detection Kit (S7101; Millipore, Billerica,
MA, USA) according to the manufacturer’s
instructions.

Zhang X., Zhu J., Liu F., Li Y., Chandra A., Levin L.S., Beier F., Enomoto-Iwamoto M, & Qin L. (2014). Reduced EGFR signaling enhances cartilage destruction in a mouse osteoarthritis model. Bone Research, 2, 14015-.

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Antibody Characterization for HIF Signaling

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Anti-HA antibody was obtained from Santa Cruz Biotechnology. Anti-HIF1α (610958) was obtained from BD Transduction Laboratories. Anti–HIF2α was from Novus Biologicals (NB100-122). Anti-glucose transporter type 1 (GLUT1) antibody was obtained from Abcam. Anti-DNMT1 (CST 5119 S) was purchased from Cell Signalling Technology. Anti- vinculin (V9264) was purchased from Sigma.

Robinson C.M., Lefebvre F., Poon B.P., Bousard A., Fan X., Lathrop M., Tost J., Kim W.Y., Riazalhosseini Y, & Ohh M. (2018). Consequences of VHL Loss on Global DNA Methylome. Scientific Reports, 8, 3313.

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Comparative Analysis of HIF-1α and HIF-2α

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The expression of HIF-1α and HIF-2α was compared in non-pigmented and pigmented melanoma cells, with Caki cells as a positive control for HIF-2α [60 (link)], and Hif1a wild type (WT) and knockout (KO) mammary tumor epithelial cells (MTECs) derived from the polyoma virus middle T transgenic mouse (MMTV-PyMT) [61 (link)] as controls for HIF-1α. Cells were grown to 80% confluence and harvested after culture for 6 hours at either normoxia (ambient air; 5% CO₂) or hypoxia (0.5% O₂, 5% CO₂). Cell extracts were prepared using a modified RIPA buffer to produce a whole cell extract (WCE) comprised of cytoplasmic and nuclear proteins. Insoluble material remaining after preparation of WCE was then re-extracted in high-salt (HS) (400 mM NaCl) buffer containing the deubiquitinase inhibitor N-ethylmaleimide (NEM, 0.5 μM) to enrich for nuclear proteins. HS-WCE was resolved on 3% to 8% Tris-acetate gels (10 μg/lane; Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in 5% milk prior to addition of anti-HIF1A (1:5,000, Novus Biologicals, NB100-479) or anti-HIF2A (1:2,000, Novus Biologicals, NB100-122) primary antibodies, which were incubated overnight at 4°C followed by incubation in anti-rabbit igG-HRP (Jackson Laboratories, 1;40,000) for 45 minutes and detection using enhanced chemiluminescence (ECL).

Slominski A., Kim T.K., Brozyna A.A., Janjetovic Z., Brooks D., Schwab L., Skobowiat C., Jozwicki W, & Seagroves T. (2014). The role of melanogenesis in regulation of melanoma behavior: Melanogenesis leads to stimulation of HIF-1α expression and HIF-dependent attendant pathways. Archives of biochemistry and biophysics, 563, 79-93.

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Hypoxia Signaling Pathway Profiling

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Lung tissue was frozen with liquid nitrogen and embedded in Optimal cutting temperature compound (4583, Sakura) and sectioned at 10 μm thickness, followed by immediate fixation in 96% ice‐cold ethanol (01396, Histolab) for 30 minutes followed by 3× 5 minute washing in PBS (14190‐144, Gibco). Tissue staining was then performed using BlockAid^™ (B10710, Invitrogen), followed by overnight incubation in 4°C with primary antibodies against HIF1a (ab82832, abcam) or HIF2a (NB100‐122, Novus) diluted 100 times. Detection was performed by staining with anti‐Rabbit IgG Alexa Fluor Plus 680 (A32802, Invitrogen) at room temperature for 1 hour followed by washing with PBS and mounting with ECTASHIELD^® Antifade Mounting Medium with DAPI (H1200, Vector Laboratories).

Gojkovic M., Darmasaputra G.S., Veliça P., Rundqvist H, & Johnson R.S. (2020). Deregulated hypoxic response in myeloid cells: A model for high‐altitude pulmonary oedema (HAPE). Acta Physiologica (Oxford, England), 229(2), e13461.

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