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.

We offer a wide range of antibody labeling technologies and dyes for antibody labeling that can be used to monitor internalization.


pHrodo iFL technology for monitoring internalization of antibodies

Fluorophores whose fluorescence is unaffected by the environment (e.g., Invitrogen Alexa Fluor dyes), require the use of quenchers in internalization. In contrast, pHrodo iFL dyes are nonfluorescent at neutral pH and become brightly fluorescent in the acidic environment as they are internalized, thus eliminating the need for a quencher.

pHrodo iFL 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.

pH response profi les of trastuzumab conjugates prepared with amine-reactive Invitrogen pHrodo iFL Red and pHrodo iFLGreen reagents
pH response profiles of trastuzumab conjugates prepared with amine-reactive Invitrogen pHrodo iFL Red and pHrodo iFLGreen reagents. These conjugates will be minimally fluorescent at a neutral pH outside of cells, but they will become brightly fluorescent upon internalization into cells.

pHrodo iFL antibody labeling methods

The pHrodo iFL Green and pHrodo iFL Red reactive dyes are modified forms of the original pHrodo dyes. They have been optimized for solubility making them useful for labeling antibodies that may otherwise precipitate out of solution during conjugation. These dyes have been combined with three different antibody labeling technologies to suit your needs for your internalization assay.

Site Specific SiteClick antibody-labeling technology

The SiteClick antibody labeling system allows simple and gentle site-specific attachment of detection molecules to heavy chain N-linked glycans—far from the antigen-binding domain—providing excellent reproducibility from labeling to labeling and from antibody to antibody regardless of isotype or host species. Antibodies can be modified to incorporate an azido moiety on their sugar residues making them click ready for attachment with our range of Click-iT sDIBO Alkynes that are available with pHrodo iFL as well as a range of Alexa Fluor dyes , biotin and reactive forms for attachment of a molecule of your choice.

High-content analysis of HER2-positive cells incubated with SiteClicked  pHrodo iFL Red–labeled Trastuzumab (false colored green) and Hoescht 33342 counter stain
High-content analysis of HER2-positive cells incubated with SiteClicked pHrodo iFL Red–labeled Trastuzumab (false colored green) and Hoescht 33342 counter stain.

Zenon antibody labeling with pHrodo iFL dyes for rapid screening

The Zenon Antibody Labeling Kits provide a means of very rapidly labeling IgG antibodies (in ~10 minutes) making it a useful tool for antibody internalization screening. The Zenon labeling method employs  Fab fragments coupled either to pH-sensitive pHrodo iFL dyes or to classic Alexa Fluor dyes. These Fab fragments are directed against the Fc portion of the IgG antibody, leaving the antigen recognition site of the target antibody intact and free from obstruction, while providing a consistent degree of labeling. Zenon technology can label as little as 1 μg of antibody, and unlike traditional labeling methods using amine- or thiol-reactive labels, the Zenon antibody labeling is compatible with BSA and other stabilizing proteins.

The Zenon pHrodo iFL labeling reagents
The Zenon pHrodo iFL labeling reagents can be complexed with human or mouse IgG to provide rapid labeling for fast, scalable screening of antibody internalization for many antibody samples.

Traditional pHrodo iFL Red and Green amine-reactive dyes and labeling kits

pHrodo iFL products are available for traditional antibody labeling of available lysines on antibodies. These amine reactive pHrodo iFL STP esters are available in green and red versions, as either stand-alone packages or microscale protein labeling kits that contain everything you need to label and purify your protein or antibody. 


Antibody labeling for internalization studies

Name Labeling scale SKU
Zenon pHrodo iFL Green Mouse IgG Labeling Kit Up to four  96 well plates Z25609
Zenon pHrodo iFL Green Mouse IgG Labeling Kit Up to four  96 well plates Z25610
Zenon pHrodo iFL Red  Mouse IgG Labeling Kit Up to four  96 well plates   Z25611
Zenon pHrodo iFL Red Human IgG Labeling Kit Up to four  96 well plates Z25612
Name Labeling scale SKU
Zenon pHrodo iFL Green Mouse IgG Labeling Kit Up to four  96 well plates Z25609
Zenon pHrodo iFL Green Mouse IgG Labeling Kit Up to four  96 well plates Z25610
Zenon pHrodo iFL Red  Mouse IgG Labeling Kit Up to four  96 well plates   Z25611
Zenon pHrodo iFL Red Human IgG Labeling Kit Up to four  96 well plates Z25612

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