Antibodies specific for cell surface receptors induce receptor-mediated endocytosis when they bind. They are commonly studied as vehicles for targeted delivery of drugs, toxins, enzymes or DNA for therapeutic applications.

Life Technologies offers a wide range of dyes for antibody labeling that can be used in wash or quench-based assays to monitor internalization. Antibody internalization can also be monitored in a no-wash assay using pHrodo™ dyes, which become fluorescent only after acidification.

 


No-wash assays

The most convenient, no-wash assay format uses pHrodo™ Red or pHrodo™ Green to allow discrimination of stages in antibody internalization from early endosome formation with no quench or wash required.

pHrodo™ dyes are essentially non-fluorescent at neutral pH and exhibit increasing signal with a red or green readout respectively as the dye is internalized and pH decreases. The increase in fluorescent signal can be used to monitor progression in the endocytic pathway.


Monitoring antibody internalization and acidification.

The amine-reactive pHrodo™ Red, succinimidyl ester was used to label lysine residues on Goat anti Mouse IgG at molar ratios of 10 and 20. Then the pH response was tested for and found to have the expected pKa of 6.8.


Antibody labeling

Antibodies specific for cell surface receptors induce receptor-mediated endocytosis when they bind. They are commonly studied as vehicles for targeted delivery of drugs, toxins, enzymes, or DNA for therapeutic applications.

Life Technologies offers a wide range of dyes for antibody labeling kits that can be used in wash or quench-based assays to monitor internalization. Antibody internalization can be monitored in a no-wash assay using pHrodo™ dyes which become fluorescent only after acidification.

Antibody labeling

Two-color confocal image of a human epidermal whole mount.

ß1 integrin was visualized with the monoclonal antibody P5D2 labeled with the green-fluorescent Alexa Fluor® 488 dye using the Alexa Fluor® 488 Monoclonal Antibody Labeling Kit. a6 integrin was labeled using a mouse monoclonal antibody visualized with Alexa Fluor® 594 Goat Anti–Mouse IgG antibody and pseudocolored blue. Image contributed by Uffe Birk Jensen, University of Aarhus, Denmark.


pHrodo™ reactive dyes selection guide

 
Reactivity
Labels amine groups 
Labels amine groups 
Labels thiol groups 
 Labels thiol groups 
Labels amine groups  
Labels biotin conjugates  
Common filter set
TRITC
FITC
TRITC
FITC
TRITC
TRITC
Labels
pHrodo™ Red
pHrodo™ Green
pHrodo™ Red
pHrodo™ Green
pHrodo™ Red
pHrodo™ Red
Ex/Em (nm)
560/585
509/533
560/585
509/533
560/585
560/585
Signal-to-noise ratio
Photostability
Format
1 mg
500 µg
1 mg
1 mg
1 kit
1 mg
Cat. No.