Ab63913

Manufactured by Abcam

Ab63913 is a mouse monoclonal antibody that recognizes the RAGE (Receptor for Advanced Glycation End products) protein. RAGE is a multi-ligand receptor that belongs to the immunoglobulin superfamily and is involved in various cellular processes.

Automatically generated - may contain errors

Lab products found in correlation

Triton x 100, by Merck Group (7 mentions) Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (7 mentions) Sodium perborate, by Merck Group (7 mentions) Ab9071, by Abcam (7 mentions) Phosphate citrate buffer, by Merck Group (2 mentions) Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Proteosilver silver stain kit, by Merck Group (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Ab205718, by Abcam (1 mentions) Femto human dna quantification kit, by Zymo Research (1 mentions) Amersham imager, by Cytiva (1 mentions)

8 protocols using ab63913

Protein Analysis of TFF Samples

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The protein composition of TFF samples and chromatographic fractions was analyzed by SDS-PAGE using NuPAGE 4%–12% Bis-Tris Protein Gels (Invitrogen), which were stained with SimplyBlue SafeStain Coomassie Brilliant Blue solution (Invitrogen). Selected gels were further stained with ProteoSilver Silver Stain Kit (Sigma-Aldrich). Western blots were performed using iBLOT 2 Dry Blotting System (Invitrogen) and iBlot 2 Transfer Stacks, polyvinylidene fluoride (PVDF) (Invitrogen). Membranes were blocked in SuperBlock T20 (PBS) Blocking Buffer (Invitrogen). Primary antibody was Rabbit polyclonal to HIV p24 (Abcam, ab63913, 1/2,500) and secondary antibody was Goat Anti-Rabbit immunoglobulin G (IgG) H&L (horseradish peroxidase [HRP]) (Abcam, ab205718, 1/20,000). Membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Stained gels and membranes were documented using Amersham Imager 600 (GE Healthcare). DC protein assay (Bio-Rad) and HEK293 HCP ELISA kit (Cygnus) were used for protein quantification, according to the manufacturers’ instructions. HC DNA was quantified using Femto Human DNA Quantification Kit (Zymo Research), according to the manufacturer’s instructions.

Ruscic J., Perry C., Mukhopadhyay T., Takeuchi Y, & Bracewell D.G. (2019). Lentiviral Vector Purification Using Nanofiber Ion-Exchange Chromatography. Molecular Therapy. Methods & Clinical Development, 15, 52-62.

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Quantitative p24 ELISA Protocol

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ELISA plate was coated with 50 ng/well of mouse anti-p24 (ab9071, Abcam, Cambridge, MA, USA) overnight at 4 °C. Following the overnight incubation, the plate was blocked with 3% (w/v) BSA at room temperature for 2 h and washed with 0.5% (v/v) Tween in PBS. Pseudoviral stocks were lysed using 0.1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h and added to the plate overnight at 4 °C. Simultaneously, p24 protein (produced and purified as previously described) was used as a standard. The following day, the plate was washed with 0.5% PBST and a 1:5000 dilution of rabbit anti-p24 (ab63913, Abcam) was added for 2 h at room temperature. After washing with PBST to remove the unbound rabbit anti-p24 off the plate, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 h at room temperature. The plate was then extensively washed with PBST. Subsequently, a solution of 0.4 mg/mL O-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated for 30 min in the dark. Optical densities were then obtained at 450 nm in a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).

Meuser M.E., Rashad A.A., Ozorowski G., Dick A., Ward A.B, & Cocklin S. (2019). Field-Based Affinity Optimization of a Novel Azabicyclohexane Scaffold HIV-1 Entry Inhibitor. Molecules, 24(8), 1581.

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Quantification of Viral Capsid Protein p24

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An ELISA plate was coated with 50 ng of mouse anti-p24 (Abcam, ab9071) per well for 2 hours at room temperature, blocked with 3% BSA for 2 hours at room temperature and washed with PBST buffer (0.1% Tween-20 in PBS). Pseudoviral stocks were lysed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 hour and added to the plate overnight at 4°C. Simultaneously, p24 protein (produced and purified as described in below) was added for the generation of a standard curve. Following the overnight incubation, the plate was washed with PBST buffer and 1:5000 dilution of rabbit anti-p24 (Abcam, ab63913) was added for 2 hours at room temperature. After washing the unbound rabbit anti-p24 off the plate with PBST buffer, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 hour at room temperature. The plate was then extensively washed with PBST buffer. Subsequently, a solution of 0.4 mg/ml o-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated in the dark for 30 minutes. Optical densities were then obtained at 450 nm in a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).

Xu J.P., Francis A.C., Meuser M.E., Mankowski M., Ptak R.G., Rashad A.A., Melikyan G.B, & Cocklin S. (2018). Exploring Modifications of an HIV-1 Capsid Inhibitor: Design, Synthesis, and Mechanism of Action. Journal of drug design and research, 5(2), 1070.

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ELISA Detection of HIV-1 p24 Antigen

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Mouse anti-p24 (Abcam, ab9071) coated an ELISA plate (50 ng/well) overnight at 4 °C. Following the overnight incubation, the plate was blocked for 2 h with 3% (w/v) room temperature BSA and washed with 0.5% (v/v) Tween in PBS. Pseudoviral stocks were lysed using 0.1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h and added to the plate overnight at 4 °C. Simultaneously, p24 protein (expressed and purified as previously described) was used as a standard. The next day, 0.5% PBST was used to wash the plate. Subsequently, rabbit anti-p24 (Abcam, ab63913) was added in a 1:5000 dilution at room temperature. After 2 h, PBST was used to wash unbound rabbit anti-p24. Next, goat anti-rabbit-HRP was added in a 1:5000 dilution at room temperature for one hour. PBST was used to thoroughly wash the plate. Subsequently, a solution of 0.4 mg/mL o-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added. This was incubated in the dark for 30 min. Using Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific), the optical densities were measured at 450 nm.

Karadsheh R., Meuser M.E, & Cocklin S. (2020). Composition and Orientation of the Core Region of Novel HIV-1 Entry Inhibitors Influences Metabolic Stability. Molecules, 25(6), 1430.

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HIV Pseudovirus Quantification by ELISA

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An ELISA plate was coated with 50 ng of mouse anti-p24 (Abcam, ab9071) per well for 2 hours at room temperature, blocked with 3% BSA for 2 hours at room temperature and washed with PBST buffer (0.1% Tween-20 in PBS). Pseudovirus stocks were lysed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 hour and added to the plate overnight at 4°C. Simultaneously, p24 protein was added for the generation of a standard curve. Following the overnight incubation, the plate was washed with PBST buffer, and 1:5000 dilution of rabbit anti-p24 (Abcam, ab63913) was added for 2 hours at room temperature. After washing the unbound rabbit anti-p24 off the plate with PBST buffer, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 hour at room temperature. The plate was then extensively washed with PBST buffer. Subsequently, a solution of 0.4 mg/ml o-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated in the dark for 30 minutes. Optical densities were then obtained at 450 nm in a Multiskan^™ GO Microplate Spectrophotometer (Thermo Scientific).

Sun L., Dick A., Meuser M.E., Huang T., Zalloum W.A., Chen C.H., Cherukupalli S., Xu S., Ding X., Gao P., Kang D., De Clercq E., Pannecouque C., Cocklin S., Lee K.H., Liu X, & Zhan P. (2020). Design, Synthesis, and Mechanism Study of Benzenesulfonamide-containing Phenylalanine Derivatives as Novel HIV-1 Capsid Inhibitors with Improved Antiviral Activities. Journal of medicinal chemistry, 63(9), 4790-4810.

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ELISA Quantification of Viral p24

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ELISA plate was coated with 50 ng of mouse anti-p24 (Abcam, ab9071, Cambridge, MA, USA) for overnight at 4 °C, blocked with 3% BSA for 2 h at room temperature, and washed with 0.5% Tween in PBS. Pseudoviral stocks were lysed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h and added to the plate overnight at 4 °C. Simultaneously, p24 protein (produced, purified, and validated as described above) was added for the generation of a standard curve. Following the overnight incubation, the plate was washed with PBST and 1:5000 dilution of rabbit anti-p24 (Abcam, ab63913) was added for 2 h at room temperature. After washing the unbound rabbit anti-p24 off the plate with PBST, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 h at room temperature. The plate was then extensively washed with PBST. Subsequently, a solution of 0.4 mg/mL O-phenylenediamine in a phosphate–citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated in the dark for 30 min. Optical densities were then obtained at 450 nm in a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).

Meuser M.E., Murphy M.B., Rashad A.A, & Cocklin S. (2018). Kinetic Characterization of Novel HIV-1 Entry Inhibitors: Discovery of a Relationship between Off-Rate and Potency. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(8), 1940.

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Quantitative HIV-1 p24 ELISA Protocol

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ELISA plate was coated with 50ng of mouse anti-p24 (Abcam, ab9071) overnight at 4°C, blocked with 3% BSA for 2 h at room temperature and washed with 0.5% Tween in PBS. Pseudoviral stocks were lysed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 h and added to the plate overnight at 4°C. Simultaneously, p24 protein (produced, purified, and validated as described above) was added for the generation of a standard curve. Following the overnight incubation, the plate was washed with PBST and 1:5000 dilution of rabbit anti-p24 (Abcam, ab63913) was added for 2 h at room temperature. After washing the unbound rabbit anti-p24 off the plate with PBST, goat, anti-rabbit-HRP at a 1:5000 dilution was added for 1 h at room temperature. The plate was then extensively washed with PBST. Subsequently, a solution of 0.4mg/ml o-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated in the dark for 30 min. Optical densities were then obtained at 450 nm in a Multiskan^™ GO Microplate Spectrophotometer (Thermo Scientific).

Meuser M.E., Reddy P.A., Dick A., Maurancy J.M., Salvino J.M, & Cocklin S. (2021). Rapid optimization of the metabolic stability of an HIV-1 capsid inhibitor using a multistep computational workflow. Journal of medicinal chemistry, 64(7), 3747-3766.

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ELISA for Quantifying HIV-1 p24 Antigen

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An ELISA plate was coated with 50 ng of mouse anti-p24 (Abcam, ab9071) per well for 2 hours at room temperature, blocked with 3% BSA for 2 hours at room temperature and washed with PBST buffer (0.1% Tween-20 in PBS). Pseudovirus stocks were lysed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 hour and added to the plate overnight at 4°C. Simultaneously, p24 protein was added for the generation of a standard curve. Following the overnight incubation, the plate was washed with PBST buffer and 1:5000 dilution of rabbit anti-p24 (Abcam, ab63913) was added for 2 hours at room temperature. After washing the unbound rabbit anti-p24 off the plate with PBST buffer, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 hour at room temperature. The plate was then extensively washed with PBST buffer. Subsequently, a solution of 0.4 mg/ml o-phenylenediamine in a phosphate-citrate buffer with sodium perborate (Sigma-Aldrich) was added and incubated in the dark for 30 minutes. Optical densities were then obtained at 450 nm in a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).

Sun L., Huang T., Dick A., Meuser M.E., Zalloum W.A., Chen C.H., Ding X., Gao P., Cocklin S., Lee K.H., Zhan P, & Liu X. (2020). Design, Synthesis and Structure-Activity Relationships of 4-Phenyl-1H-1,2,3-Triazole Phenylalanine Derivatives as Novel HIV-1 Capsid Inhibitors with Promising Antiviral Activities. European journal of medicinal chemistry, 190, 112085.

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