Alexafluor 546 carboxylic acid succinimidyl ester

Manufactured by Thermo Fisher Scientific

AlexaFluor 546 carboxylic acid succinimidyl ester is a fluorescent dye that can be used to label proteins and other biomolecules. The dye has an absorption maximum at 556 nm and an emission maximum at 573 nm, making it suitable for a variety of fluorescence-based applications.

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Lab products found in correlation

Alexa fluor 546, by Thermo Fisher Scientific (1 mentions) Alexa fluor 546 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Pd 10 desalting column, by GE Healthcare (1 mentions) Anti tgf β rii, by Santa Cruz Biotechnology (1 mentions) Synergy 2 plate reader, by Agilent Technologies (1 mentions)

2 protocols using alexafluor 546 carboxylic acid succinimidyl ester

Fluorescent Labeling of Wnt5a for Internalization

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Purified Wnt5a (40 pmol) was fluorescently labelled with AlexaFluor 546 carboxylic acid succinimidyl ester (200 pmol; Life Technologies, Carlsbad, CA) in 200 mM sodium hydrogen carbonate [pH 7.4] for 3 h at 4°C. After quenching of excess AlexaFluor 546 with 300 mM hydroxylamine, labelled Wnt5a (Wnt5a*) was used for a receptor internalization assay. The degree of labelling of Wnt5a with AlexaFluor 546 was estimated by comparison to AlexaFluor 546-conjugated streptavidin (4 mol of AlexaFluor 546 conjugated to 1 mol of streptavidin, Life Technologies) using a fluorescence image analyzer (Tyhoon, GE). About 3.5 mol of AlexaFluor 546 was conjugated to 1 mol Wnt5a and Wnt5a* was used for the internalization assay.

Shojima K., Sato A., Hanaki H., Tsujimoto I., Nakamura M., Hattori K., Sato Y., Dohi K., Hirata M., Yamamoto H, & Kikuchi A. (2015). Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. Scientific Reports, 5, 8042.

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FRET Characterization of BMPR1 and Iβ1 Interaction

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To investigate the association of BMPR1 and Iβ1, we performed Förster Resonance Energy Transfer (FRET)^{23 (link), 27 (link)}. Briefly, the donor antibody (anti Iβ1 (SantaCruz)) was labeled with Alexa Fluor^® 488 Carboxylic Acid, Succinimidyl Ester (Life Technologies) (Iβ1-488) and the acceptor antibody (anti TGF-β RII (SantaCruz)) was labeled with Alexa Fluor^® 546 Carboxylic Acid, Succinimidyl Ester (Life Technologies) (BMPRI-546) in a 2.25:1 molar ratio of antibody:dye overnight at 4°C. Labelled antibody was purified with size exclusion chromatography using PD-10 Desalting Columns (GE Healthcare). Rat MSCs were plated on pre-soaked compliant (5 MPa) and rigid (266 MPa) PUR films. After 24 hours of culture, cells were fixed with 10% Formaldehyde in PBS and stained overnight at 4° with Iβ1-488 and Iβ1-488 + BMPRI-546 (1 ug/1×10⁶ cells). FRET experiments were performed on a BioTek Synergy 2 plate reader using excitation filter 485/20 and emission filter 530/35.

Guo R., Lu S., Merkel A.R., Sterling J.A, & Guelcher S.A. (2016). Substrate Modulus Regulates Osteogenic Differentiation of Rat Mesenchymal Stem Cells through Integrin β1 and BMP Receptor Type IA. Journal of materials chemistry. B, Materials for biology and medicine, 4(20), 3584-3593.

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