Ab2771

Manufactured by Abcam

Ab2771 is a primary antibody product from Abcam. It is designed for use in various immunoassay applications.

Automatically generated - may contain errors

Lab products found in correlation

Prism 8, by GraphPad (2 mentions) Nupage 4 12 bis tris, by Thermo Fisher Scientific (1 mentions) Image lab version 6, by Bio-Rad (1 mentions) Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions) Nupage loading buffer, by Thermo Fisher Scientific (1 mentions) Phosstop, by Roche (1 mentions) Iblot dry transfer system, by Thermo Fisher Scientific (1 mentions) Ab155102, by Abcam (1 mentions) Ab32028, by Abcam (1 mentions) Ab109268, by Abcam (1 mentions) Ab32529, by Abcam (1 mentions) Ab60948, by Abcam (1 mentions) Ab32024, by Abcam (1 mentions) β actin, by Merck Group (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Ab81283, by Abcam (1 mentions) Amersham ecl, by Cytiva (1 mentions) Ab23981, by Abcam (1 mentions) Ab209484, by Abcam (1 mentions)

4 protocols using ab2771

Quantifying Protein Expression in Immune Cells

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Patient PBMCs were lysed in RIPA buffer supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche) on ice. Protein concentration was determined using DC protein assay (Bio-Rad Laboratories) and samples prepared in 1x NuPage loading buffer (Invitrogen™) with 1x NuPage Sample Reducing Agent (Invitrogen™). Samples were run on NuPage Bis-Tris 4-12% (Invitrogen™) and NuPage Tris-Acetate 3–8% (Invitrogen™) gels and transferred using iBlot dry transfer system (Invitrogen™). Membranes were blocked 1h at RT using 5% bovine serum albumin or milk in PBSt 0.1% Tween-20 and primary antibody incubated over-night at 4°C, ENO1 (Abcam, #ab155102), ENO3 (Abcam, #ab126259), HIF-1α (BdBioscience, #610959), Akt (Abcam, #ab2771), Akt(S473) (Abcam, #ab81283), mTOR (Abcam, #ab32028), mTOR(S2448) (Abcam, #ab109268), S6K1 (Abcam, #ab32529), S6K1(T389 + T412) (Abcam, #ab60948), 4EBP1 (Abcam, #ab32024), 4EBP1(T37) (Abcam, #ab75767), or β-Actin (Sigma-Aldrich, #A5441). The secondary antibody (Dako, Aglient) was incubated 1h at RT prior detection using Amersham ECL/ECL select (GE Healthcare). Relative protein quantification was analysed using ImageLab version 6.0.1 (Bio-Rad Laboratories), results analysed using Mann-Whitney U-test or unpaired t-test and visualized using Prism 8.4.3 (GraphPad Software) (significance level, p<0.05).

Akusjärvi S.S., Ambikan A.T., Krishnan S., Gupta S., Sperk M., Végvári Á., Mikaeloff F., Healy K., Vesterbacka J., Nowak P., Sönnerborg A, & Neogi U. (2021). Integrative proteo-transcriptomic and immunophenotyping signatures of HIV-1 elite control phenotype: A cross-talk between glycolysis and HIF signaling. iScience, 25(1), 103607.

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Bone Development in miR-29cb2 Knockout Mice

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miR-29cb2^−/− mice were sacrificed at the ages of 6 and 16 weeks. The femurs were collected, fixed with 4% PFA, decalcified and cut into 6 μm thick transverse sections. HE (Servicebio), an anti-Runx-2 antibody (rabbit; 1:1000; ab23981; Abcam), Anti-Sp7 / Osterix antibody (Rabbit; 1:1000; ab209484; Abcam) and Trap (Servicebio) were used for morphological examination. anti-HIF-1β (rabbit; 1:1000; ab2771; Abcam) and anti-HIF-3α (rabbit; 1:14000; NBP1-03155; Novus) were used for target examination. Digital images showing each antigen were acquired and evaluated using Image-Pro Plus software.

Ouyang L., Sun Y., Lv D., Peng X., Liu X., Ci L., Zhang G., Yuan B., Li L., Fei J., Ma J., Liu X, & Liao Y. (2021). miR-29cb2 promotes angiogenesis and osteogenesis by inhibiting HIF-3α in bone. iScience, 25(1), 103604.

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Western Blot Analysis of HIF-1 Signaling

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Cells were harvested, rinsed with PBS and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA,1 mM MgCl₂, 0.5% NP-40, 1 mM Na₃VO₄, 1 mM NaF, protease inhibitors cocktail). Cell lysates were separated on SDS–polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories) and immunoblotted using the following primary antibodies. Rabbit anti-HIF-1α (1:1,000, GTX127309), anti-GST (1:5,000, GTX110736), anti-lamin B2 (1:5,000, GTX109894) and anti-tubulin (1:5,000, GTX112141) antibodies, as well as mouse monoclonal anti-HIF-1α antibody (1:1,000, GTX628480), were purchased from GeneTex. Mouse monoclonal antibodies recognizing β-actin (1:5,000, A2228) were purchased from Sigma. Mouse monoclonal HIF-1β antibody [2B10] (1:2,000, ab2771) were purchased from Abcam. Uncropped scans of the blots and gels are shown in Supplementary Fig. 19 in the Supplementary Information section.

Shih J.W., Chiang W.F., Wu A.T., Wu M.H., Wang L.Y., Yu Y.L., Hung Y.W., Wang W.C., Chu C.Y., Hung C.L., Changou C.A., Yen Y, & Kung H.J. (2017). Long noncoding RNA LncHIFCAR/MIR31HG is a HIF-1α co-activator driving oral cancer progression. Nature Communications, 8, 15874.

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Quantifying Hypoxia-Induced Immune Responses

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PBMCs (250,000/coverslip) were added in 100μL PBS on poly-L-lysine pre-coated coverslips (BioCoat™) and left to adhere 30 min at 37°C. After attachment coverslips were washed in PBS and fixed 10 min at RT using 4% PFA. Cells were blocked using 3% BSA and antibody detection of CD4 (R&D systems, #AF-379-NA) and Alexa 647 conjugated CD8 (Abcam, #ab196193) performed prior permeabilization 10 min using 0.2% Triton x-100 and subsequent primary antibody incubation targeting HIF-1α (Novus Biologicals, #NB-100-449) and HIF-1β (Abcam, #ab2771). Secondary antibodies, Alexa Fluor 488 (Invitrogen, #A32731), Alexa Fluor 568 (Invitrogen, #A-11004), and Alexa Fluor 647 (Invitrogen, #A-21447) were incubated 1h at RT. Cells were counterstained using DAPI and mounted with Prolong Gold Antifade reagent (Thermofisher). Images were acquired using Nikon single point scanning confocal microscope with 60x/1.4 oil objective. All samples were analysed in two technical replicates and detection threshold was set using secondary antibody controls. Image analysis was performed in Imaris (Bitplane) using detection of Nucleus, Cell and Vesicles detection according to pipeline in Figures S4A and S4B. Graphical representation was performed in Imaris and statistical analysis was performed using Mann-Whitney U-test in Prism 8.4.3 (GraphPad Software) (significance level, p<0.05).

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