400 ng of each of pcDNA3-gH (full-length), pcDNA3-gL-FLAG and pcDNA3-gO-8his were transfected in to Expi293F cells at 2 × 106 / ml using Expifectamine. Transfection Enhancers were added 22 h later, and cells were harvested 46 h post-transfection. Cells were washed with PBS-BSA and incubated with PDGFRα-Fc (purified as described) or PDGFRβ-Fc (R&D Systems) for 40 minutes on ice. Cells were then washed 3 times with PBS-BSA and stained with anti-FLAG M2-Cy3 (Sigma), chicken anti-HIS-FITC (polyclonal, Immunology Consultants Laboratory), and anti-human IgG-APC (clone HP6017, BioLegend) for 30 minutes on ice. Cells were washed three times with PBS-BSA and analyzed by flow cytometry. To compare data across different experiments, the change in ΔMFU for each condition was normalized to the ΔMFU at the maximum concentration of WT sPDGFRα-Fc: Relative binding = (MFUsample−MFUbackground) / (MFUmax WT − MFUbackground).
Anti flag m2 cy3
Manufactured by Merck Group
Sourced in United Kingdom
The Anti-FLAG M2-Cy3 is a fluorescently-labeled antibody used for detection and purification of FLAG-tagged proteins. The Cy3 fluorescent dye is conjugated to the Anti-FLAG M2 antibody, providing a sensitive and specific tool for visualizing and isolating FLAG-tagged proteins in various applications.
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Lab products found in correlation
Pdgfrβ fc, by R&D Systems (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti ha high affinity, by Merck Group (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Alexa flour 488, by Beyotime (1 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti nestin, by Abcam (1 mentions)
Anti clic1, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Abcam (1 mentions)
Anti βiii tubulin, by Merck Group (1 mentions)
Iaa94, by Merck Group (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti rb, by BD (1 mentions)
Metformin, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
5 protocols using anti flag m2 cy3
1
PDGFR Binding Assay by Flow Cytometry
2
Retina Flat Mount and Immunostaining Protocol
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Enucleated eyes were processed for cryosectioning or flat mounts. Tissue sampling for cryosections was conducted as previously described.25 (link) For flat mounts, retinas were carefully dissected and flattened onto nitrocellulose membranes with the RGC layer facing upwards. Retinas were fixed for 30 minutes with 4% paraformaldehyde at room temperature and then transferred to PBS at 4°C (for up to 3 days). The retinas were then carefully teased off the membranes and treated with ascending concentrations of sucrose (10%, 20%, and 30%) each for 10 minutes, followed by 3 freeze/thaw cycles on dry ice and room temperature, respectively. The retina flat mounts were mounted onto Superfrost slides (Fisher Scientific, Waltham, MA) in 30% sucrose and placed at –80°C until staining. Hematoxylin and eosin staining was performed according to standard protocols. Immunostaining was performed with anti-FLAG M2-Cy3 (1:100, Sigma, St. Louis, MO), anti-HA high affinity (1:100, Sigma), anti-RNA binding protein with multiple splicing (RBPMS; 1:100, Phosphosolutions, Aurora, CO) and anti-GFP (1:200, Invitrogen, Carlsbad, CA). All primary antibodies were incubated for 2 days. Secondary antibodies conjugated to Alexa 488 (Invitrogen) and Cy3 (Jackson ImmunoResearch, West Grove, PA) fluorophores were used at 1:500 and incubated at room temperature for 1 hour.
3
Visualization of TUBB8 Localization
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Twenty-four hours after seeding, HEK 293T cells were transiently transfected with WT and mutant C-terminally Myc-DDK (FLAG)–tagged TUBB8 plasmids. The cells were gently washed three times using PBS 48 h after transfection. Then, 2% paraformaldehyde was applied to fix the cells. Permeabilization was performed with 0.5% Triton X-100 for 30 min at room temperature after being washed with PBS three times. Nonspecific binding was blocked by 5% BSA at room temperature for 1 h. The cells were then incubated in the dark at 4°C overnight with diluted antibodies: anti-FLAG M2-Cy3 (sigma, #A9594, 1:500), anti-α tubulin (cell signaling technology, #5063S, Alexa flour 488 conjugated, 1:250), and DAPI (Beyotime, #C1002, 1:1000). The cells were washed three times using 0.5% Triton X-100, adding 0.5% Tween-20 before mounting. The confocal images were taken using Leica TCS SP8 confocal laser scanning microscope.
4
Metformin and IAA94 in Cell Characterization
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Metformin (1,1-dimethylbiguanide-hydrochloride) and IAA94 (indanyloxyacetic acid 94) were from Sigma-Aldrich (Milano, Italy).
Antibodies: anti-CLIC1, Santa Cruz (Dallas, USA); anti-GFAP, anti-Nestin, and anti-N cadherin, Abcam (Cambridge, UK); anti-α-tubulin, anti-β-III tubulin, anti-FLAG M2-Cy3 and anti-FLAG M2, Sigma-Aldrich (Milano, Italy); anti-Olig2 and anti-Sox2, Millipore (Vimodrone, Italy); anti-Rb, BD Biosciences (Milano, Italy); Alexa Fluor 488 anti-mouse and Alexa Fluor 546 anti-phalloidin, Invitrogen (Carlsbad CA, USA); DyLight 459-goat anti-rabbit IgG, Jackson Immunoresearch (West Grove, USA).
Antibodies: anti-CLIC1, Santa Cruz (Dallas, USA); anti-GFAP, anti-Nestin, and anti-N cadherin, Abcam (Cambridge, UK); anti-α-tubulin, anti-β-III tubulin, anti-FLAG M2-Cy3 and anti-FLAG M2, Sigma-Aldrich (Milano, Italy); anti-Olig2 and anti-Sox2, Millipore (Vimodrone, Italy); anti-Rb, BD Biosciences (Milano, Italy); Alexa Fluor 488 anti-mouse and Alexa Fluor 546 anti-phalloidin, Invitrogen (Carlsbad CA, USA); DyLight 459-goat anti-rabbit IgG, Jackson Immunoresearch (West Grove, USA).
5
Fluorescent Receptor Localization Assay
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Cells were co-transfected with FRET-based sensors to monitor downstream signaling and the wild type or the BRET-biosensor receptor as described above. Staining of the plasma membrane portion of the receptors was evaluated using a cell-impermeable anti HA-tag conjugated with AlexaFluor594 (Anti-HA-AlexaFluor594 ThermoFischer) or Anti-Flag® M2 conjugated with Cy3 (Anti-Flag® M2 Cy3, Sigma). The fluorescent antibodies were diluted in DMEM to a concentration of 10 µg mL−1 and incubated for 1 h at 37 °C in the 96-well plates. Subsequently, cells were rinsed three times and incubated additional 30 min with fresh DMEM.
Subsequently, the emission intensity of HEK-TSA cells were measured using the Synergy Neo2 plate reader. Therefore, cells stained with Anti-Flag® M2 Cy3 (β2ARNLuc/Halo or β2AR) were excited using a 540/20 nm excitation and the emission intensity was recorded using 590/35 nm emission filter. Fluorescence intensities of HEK-TSA cells stained with Anti-HA-AlexaFluor594 were measured using a 590/20 nm (excitation)–620/15 nm (emission) filter combination.
Subsequently, the emission intensity of HEK-TSA cells were measured using the Synergy Neo2 plate reader. Therefore, cells stained with Anti-Flag® M2 Cy3 (β2ARNLuc/Halo or β2AR) were excited using a 540/20 nm excitation and the emission intensity was recorded using 590/35 nm emission filter. Fluorescence intensities of HEK-TSA cells stained with Anti-HA-AlexaFluor594 were measured using a 590/20 nm (excitation)–620/15 nm (emission) filter combination.
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