pH-sensitive lgG labeling reagents (Deep Red)

Antibody internalization labeling probe. It can bind to candidated antibodies to produce soluble complex that only fluoresce in acidic environments for the study of antibody internalization.
The pH-sensitive Labeling probe binds to your primary antibodies via Fc binding protein to a pH-dependent fluorescent molecular. This fluorescent complex reporter will increase intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell. The internalized antibodies are detected by measuring fluorescence intensity of the cells.

This product can becan be used for human IgG1, IgG2, IgG3, IgG4, rabbit IgG, mouse IgG1, IgG2a, IgG2b and IgG3 and can be detected with Flow cytometry Cy5 filter.

1. Preparation of UA079026 and Antibody Incubation

1.1 Reconstitution of UA079026

- Dissolve the lyophilized UA079026 in deionized water according to the specified dissolution method.

1.2 Preparation of 4X Antibody Working Solution

- Prepare a sufficient volume of 4X working solution of the test antibody using cell culture medium (four times the test concentration, which is determined based on preliminary flow cytometry conditions). For example, if 2μg/ml is a good starting test concentration for the antibody, then the 4X working solution would be 8μg/ml.

1.3 Preparation of 4X UA079026 Working Solution

- Prepare a sufficient volume of 4X working solution of UA079026 using cell culture medium. The molar ratio of the test antibody to UA079026 should be 1:2.

1.4 Mixing and Incubation

- Mix the 4X test antibody working solution and the 4X UA079026 working solution in a 1:1 volume ratio. Incubate at room temperature in the dark for 15min- 1 hour to obtain the 2X Ab-UA079026 complex working solution.

2. Incubation of Ab-UA079026 Complex with Cells

2.1 Cell Preparation

- Collect and wash the cells. Adjust the cell concentration using complete culture medium. For suspension cells, adjust to a concentration of 1-2×10⁵ cells/mL. For adherent cells, adjust to a concentration of 0.5-1×10⁵ cells/mL. Add 100μL of cell suspension to each well of a 96-well plate.

2.2 Incubation

- Add 100μL of the 2X Ab-UA079026 complex working solution to each well. Incubate in a 37℃, 5% CO₂ incubator.

3. Flow Cytometry Detection

- After 18-24 hours of incubation(Adjustable according to the actual endocytosis time of the antibody), collect the cells for flow cytometry to detect the internalization effect of the antibody. Use the Cy5 channel for detection.