Description
Incucyte®Human Fabfluor-pH Antibody Labeling Dye for Antibody Internalization allows for a simple, effective labeling process. It was designed to quickly label Fc-containing test antibodies with a Fab fragment-conjugated pH-sensitive fluorophore. The pH-sensitive dye based system exploits the acidic environment of the lysosomes to quantify internalization of the labeled antibody. As Fabfluor labeled antibodies reside in the neutral extracellular solution (pH 7.4), they interact with cell surface specific antigens and are internalized. Once in the lysosomes, they enter an acidic environment (pH 4.5–5.5) and a substantial increase in fluorescence is observed. In the absence of expression of the specific antigen, no internalization occurs and the fluorescence intensity of the labeled antibodies remains low.
Based on immunoelectrophoresis and/ or ELISA, the antibody reacts with the Fc portion of human IgG heavy chain but not the Fab portion of human IgG. No antibody was detected against human IgM, IgA or against non-immunoglobulin serum proteins. The antibody may cross-react with other immunoglobulins from other species.
With the Incucyte®integrated analysis software, background fluorescence is minimized. These reagents have been validated for use with a number of different antibodies in a range of cell types. Together with the Incucyte®Live-Cell Analysis System, the Incucyte®Fabfluor-pH Antibody Labeling Dyes enable real-time, kinetic evaluation of antibody internalization.
Presentation, Storage and Stability
The Incucyte®Fabfluor-pH Antibody Labeling Dyes for antibody internalization are supplied as lyophilized solids in sufficient quantity to label 50 μg of test antibody, when used at the suggested molar ratio (1:3 of test antibody to labeling Fab). The lyophilized solid can be stored at 2-8° C for one year. Once re-hydrated, any unused reagent should be aliquoted and stored at -80° C for up to one year. Avoid repeated freeze-thaw cycles.
Recommended Use
The Incucyte®Fabfluor-pH Antibody Labeling Dyes are to be prepared at a stock concentration of 0.5 mg/mL by the addition of 100 μL of sterile water and triturated (centrifuge if solution not clear). The reagent may then be diluted directly into the labeling mixture with the test antibody. Do NOT sonicate the solution.
Additional protocol details can be found in the Product Guide.
