Pe donkey anti rabbit

Manufactured by BioLegend

Sourced in United States

The PE donkey anti-rabbit is a secondary antibody conjugated with the fluorescent dye Phycoerythrin (PE). It is designed to detect and bind to primary antibodies raised in rabbit, allowing for the visualization and analysis of target proteins or cells in various applications, such as flow cytometry, immunohistochemistry, and Western blotting.

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Lab products found in correlation

Sh800 cell sorter, by Sony (1 mentions) Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions) Facscalibur, by BD (1 mentions) Calphos mammalian transfection kit, by Takara Bio (1 mentions) Kaluza software, by Beckman Coulter (1 mentions) Cr3022, by Abcam (1 mentions) Facsdiva software, by BD (1 mentions) Enzyme free cell dissociation buffer, by Thermo Fisher Scientific (1 mentions) Lsrfortessa cell analyzer, by BD (1 mentions)

Close spelling variants of this product

PE Donkey anti-rabbit IgG (7 citations) PE-conjugated donkey anti-rabbit IgG (4 citations) PE donkey anti-rabbit IgG antibody (3 citations) PE donkey anti-rabbit IgG (Poly4064) (2 citations) PE-conjugated donkey anti-rabbit IgG antibody (2 citations) Donkey anti-rabbit PE antibody (2 citations) Secondary donkey anti rabbit-PE antibody (2 citations)

4 protocols using pe donkey anti rabbit

Generation of NFAT-GFP reporter and TCR-expressing 58 cell lines

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The NFAT-GFP reporter cell line (58.NFAT-GFP) was generated by transducing 58 cells with retroviral particles carrying the pSIRV-NFAT-eGFP transfer plasmid (Addgene plasmid) [35 (link)]. Five-six days after transduction, cells were plated one cell/well in 96-well plates and expanded for 2 weeks until visible colonies appeared. Colonies were then stimulated with PMA and Ionomycin (100 nM each) for 24h and analyzed by flow cytometry for GFP signal. 58.NFAT-GFP clones were selected based on having a low GFP signal in unstimulated conditions and a strong GFP signal following PMA/Ionomycin stimulation.
58.NFAT-GFP cells expressing human CD4 (58.NFAT-GFP.CD4) were generated by transducing the 58.NFAT-GFP line with retroviral particles carrying the pMX hCD4 transfer plasmid (Addgene [32 (link)]) and subsequent sorting of cells based on high CD4 expression using the SH800 cell sorter (Sony Biotechnology). Importantly, human CD4 interacts well with both human and mouse MHC class II molecules [36 (link)].
TCR expressing versions of the 58 cell line were generated by transducing 58 cells with the TCR-pMSCVII-Ametrine (hTCR-pMIA) plasmid [29 (link)], and sorting cells based on strong Ametrine signal or strong staining with anti-mouse CD3, rabbit anti-TCR alpha/TRAC Picoband polyclonal antibody (Bosterbio), and PE donkey anti-rabbit (BioLegend).

Boddul S.V., Sharma R.K., Dubnovitsky A., Raposo B., Gerstner C., Shen Y., Iyer V.S., Kasza Z., Kwok W.W., Winkler A.R., Klareskog L., Malmström V., Bettini M, & Wermeling F. (2021). In vitro and ex vivo functional characterization of human HLA-DRB1∗04 restricted T cell receptors. Journal of Translational Autoimmunity, 4, 100087.

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Multiparametric Flow Cytometry of Metabolic Markers

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Cells were fixed by suspending in PBS with 4% PFA for 15 min and then suspended in Permeabilization Buffer (PBS, 0.5% Triton X-100) for 15 min, followed by ICC-Block (PBS, 5% NGS, 0.1% Triton X-100) for 30 min. Primary antibodies were diluted in ICC-Block, incubated for 1 h, washed and then incubated in secondary antibody for 30 min before final resuspension in PBS for analysis. All incubations were done in the dark, at room temperature, and on a rocker. Flow cytometry was performed using the Attune NxT Acoustic Focusing Cytometer (ThermoFisher, Waltham, MA, USA) and accompanying software. Antibodies used include GLUT1, GLUT3, and CA-9 at 1:1000 and PE Donkey anti-Rabbit (Biolegend, San Diego, CA, USA, 406421) at 1:500. Hoechst 33342 was used at 1:1500.

Ryniawec J.M., Coope M.R., Loertscher E., Bageerathan V., de Oliveira Pessoa D., Warfel N.A., Cress A.E., Padi M, & Rogers G.C. (2022). GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors. Diagnostics, 12(3), 676.

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Spike Protein Expression Analysis

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293T Lenti-X cells (3x10⁵/well) were plated in 6-well plate and transfected with 1 µg of Spike-expressing plasmids or phCMV-VSV.G as a negative control using the CalPhos™ Mammalian Transfection Kit (Clontech). Forty-eight hours after transfection, cells were detached, counted and stained either with anti-S2 commercial antibody (Cat: 40590-T62, Sino Biological; 1:3000) followed by donkey anti-rabbit PE (Biolegend, San Diego, CA, USA; 4 µg/ml) or human monoclonal antibodies COVA2-15, COVA1-16, COVA1-18, COVA1-21 (47 (link)) (1 µg/ml) or CR3022 (Cat: ab273073, Abcam, Cambridge, UK; 5 µg/ml), followed by Goat anti-human IgG secondary AlexaFluor647 (Cat: 109-605-003, Jackson ImmunoResearch, 5 μg/ml). The expression of Spike was analyzed by flow cytometry utilizing a FACSCalibur (BD Biosciences, Milan, Italy), and the results were analyzed with Kaluza software (Beckman Coulter, Fullerton, CA, USA).

Borghi M., Gallinaro A., Pirillo M.F., Canitano A., Michelini Z., De Angelis M.L., Cecchetti S., Tinari A., Falce C., Mariotti S., Capocefalo A., Chiantore M.V., Iacobino A., Di Virgilio A., van Gils M.J., Sanders R.W., Lo Presti A., Nisini R., Negri D, & Cara A. (2023). Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles. Frontiers in Immunology, 14, 1147953.

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Dopamine Receptor Expression in Rheumatoid Arthritis

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Untreated RASF (n = 15–11) and OASF (n = 13–8) from one confluent 12 well plate were used for the staining. Cells were gently detached by using an enzyme-free cell dissociation buffer (Life Technologies, cat nr. 13151–014) and then incubated with conjugated antibodies against D1DR, D3DR or D5DR (rabbit anti-hD1DR, FITC conjugated: Antibodies online, cat nr. ABIN2173733; rabbit anti-hD3DR, Cy5 conjugated: Bioss, cat nr. BSS-BS-1743R; rabbit anti-hD5DR PE conjugated: Novus Biologicals cat nr. FAB82861P).
D2DR and D4DR were stained with antibodies targeting cytoplasmatic domains. For the intracellular staining, SF were fixed with PBS + 2% formaldehyde, then permeabilized with a specific permeabilizing solution (BD Biosciences, cat nr. 340973) prior incubation with the specific primary antibody (rabbit anti-hD2DR: Biozol, cat nr. LS-A1405; rabbit anti-hD4DR: Biorbyt, cat nr. BYT-ORB39453). After washing, cells were incubated with the specific secondary antibody (donkey anti-rabbit PE, Biolegend, cat nr. 406421).
Cells were analyzed using the FACSDiva™ Software on a LSRFortessa™ cell analyzer (BD Biosciences). Mean fluorescence intensity of the gated population (see additional figure 1) was evaluated and used for further analysis. As controls, fluorescence-minus-one (FMO) staining was performed^{29 (link)} and showed no unspecific staining (see additional figure 2).

van Nie L., Salinas-Tejedor L., Dychus N., Fasbender F., Hülser M.L., Cutolo M., Rehart S., Neumann E., Müller-Ladner U, & Capellino S. (2020). Dopamine induces in vitro migration of synovial fibroblast from patients with rheumatoid arthritis. Scientific Reports, 10, 11928.

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