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VRDye 549 Secondary Antibodies

Products

VRDye 549 secondary antibodies can be used for a variety of applications, including immunofluorescence microscopy, flow cytometry, and others. They are suitable for multiplex experiments when combined with other secondary antibodies labeled with proper fluorescent dyes and using instrumentation with appropriate excitation and detection capabilities.

Visible Fluorescence Reagents Overview

VRDye 549 Forms Available

  • Secondary antibodies
  • Labeling kits

Formulation and Recommended Dilutions

VRDye 549 secondary antibodies are supplied as purified immunoglobulin conjugates, lyophilized in phosphate-buffered saline, pH 7.4. Protect from light. Store at 4 °C prior to reconstitution.

Each vial contains 10 mg/mL BSA (free of IgG and protease) as a stabilizer and 0.01% sodium azide as a preservative, after reconstitution. Concentration is 1.0 mg/mL when reconstituted as directed. Refer to the pack insert for details on reconstitution.

Application Recommended Suggested Range
Immunofluorescence Microscopy 1:400 1:100 - 1:1,000
Flow Cytometry 1:1,000 1:200 - 1:2,000
Other User optimized

Suggested working dilutions are given as a guide only. Optimum dilutions will vary and should be determined empirically.

Absorption and Emission Spectra in 1X PBS

Properties of Visible Fluorescence NHS Ester Dyes

Dye Absmax Emmax ε(M-1cm-1) MW (g/mole) CF*
VRDye 549 550 575 150,000 1,040 0.07

*CF is a correction factor for the absorbance of the dye at Absmax to the absorbance of the protein at 280 nm.

VRDye 549 Used for Immunohistochemistry (IHC)

IHC staining of EGFR protein on F98-EGFR tumor slides. F98-EGFR tumors were snap-frozen in O.C.T. compound and sectioned at 4-μm thickness. After fixation and permeabilization, cells were incubated with rabbit anti-EGFR mAb (CST), followed by detection with VRDye 549 Goat anti-Rabbit IgG (P/N 926-54020). DAPI was used to stain the nuclei. Image acquired on Olympus IX81 microscope.
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