Fig1. The fluorescent intensity from antibody-probe protein complex internalization into cell is specific for B7-H3 on Hela cell.
pH-sensitive lgG labeling reagents (Red)
pH-sensitive lgG labeling reagents (Red)
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Product Details
Product Details
Product Specification
| Molecular Weight | 31kDa |
| Physical Appearance | Lyophilized Powder |
| Storage Buffer | PBS, PH7.4 with 5% trehalose is added as protectants |
| Reconstitution |
1. Before opening the tube cap, centrifuge the sample tube at 5000g for 3-5min at room temperature to ensure the lyophilized sample to settie down at the bottom of the tube. 2. Dissolved lyophilized protein sample in sterile water based on the recommended volume 10uL（10ug package）. 3. After adding sterile water, cover the lid and mix them bygently tapping the tube for 5-10 times. Note: Do not vortex or vigorously pipette sample. |
| Stability & Storage | · 12 months from date of receipt, lyophilized powder stored at -20 to -80℃. |
Background
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 PE filter.
Protocol
1. Incubation of UA070127 with Antibody
1.1 Reconstitute the lyophilized UA070127 powder with deionized water according to the dissolution instructions.
1.2 Prepare a sufficient volume of 4X working solution of the test antibody using cell culture medium (four times the test concentration, which should be optimized based on preliminary flow cytometry conditions). For example: 2 μg/ml may be a good starting test concentration for the antibody, so the 4X working solution would be 8 μg/ml.
1.3 Prepare a sufficient volume of 4X UA070127 working solution using cell culture medium. The molar ratio of the test antibody to UA070127 is 1:2.
1.4 Mix the 4X test antibody working solution and the 4X UA070127 working solution at a 1:1 volume ratio. Incubate at room temperature protected from light for 15min- 1h to obtain a 2X working solution of the Ab-UA070127 complex.
2. Incubation of Ab-UA070127 Complex with Cells
2.1 Cell preparation: Harvest and wash the cells, then adjust the cell concentration using complete culture medium. For suspension cells, adjust to a concentration of 1-2×10^5 cells/mL; for adherent cells, adjust to 0.5-1×10^5 cells/mL. Add 100 μL of cell suspension to each well of a 96-well plate.
2.2 Incubation: Add 100 μL of the 2X Ab-UA070127 complex working solution to each well, and culture in a 37°C, 5% CO2 incubator.
3. Flow Cytometry Analysis
After culturing for 18-24h (adjustable based on the actual internalization time of the antibody), harvest the cells and perform flow cytometry to evaluate antibody internalization. Detection should be performed using the PE channel.
Picture
Picture
Bioactivity
