pHrodo 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 and Deep Red 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 and Deep Red dyes show little to no signal at neutral pH and exhibit increasing signal with a far red, 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. pHrodo Deep Red is distinct from pHrodo iFL Green and Red in that it has a lower pKa and will not fluoresce until later in the endocytic pathway.

Fluorescent images of SKBR3 cells treated with fluorescent dyes

Low pKa pHrodo Deep Red is optimized for late endosomes and lysosomes. Herceptin (monoclonal antibody used to treat breast and stomach cancers) was labeled with pHrodo Deep Red. SKBR3 cells were treated with pHrodo Deep Red–labeled Herceptin and CellLight Early Endosome-RFP (left), CellLight Late Endosome-RFP (center), and CellLight Lysosomes-RFP (right). After overnight incubation, samples were stained with NucBlue Live nuclear dye for 30 minutes at 37˚C. Using the EVOS M7000 cell imaging system, fluorescent images were acquired and overlaid. Overlapping fluorescent signals (seen as white) demonstrate that internalized Herceptin antibody labeled with pHrodo Deep Red (magenta) fluoresces in late endosomes and lysosomes, but not early endosomes. These data suggest that the more acidified pH of 5.5–6.0 found in late endosomes and lysosomes is required to activate the pHrodo Deep Red from non-fluorescent to a deep red fluorescence.

Antibody label selection guide

  pHrodo Green iFL pHrodo Red iFL pHrodo Deep Red
Range Early endosome to lysosome Late endosome and lysosome
Common filter set FITC/GFP TRITC/RFP Cy5
Ex/Em (nm) 509/533 560/585 640/655
Signal-to-noise ratio
pKa* 6.5 6.5 5
Multiplexing Yes
Live cells Yes
Fixed cells No
Platforms Imaging, high content analysis, flow cytometry
SiteClick Site Specific - C20034 S10914
Amine-reactive labeling kit P36015 P36014 P35355
Amine-reactive reagent P36013 P36011 P35359

* Signal appears prior to environmental pH reaching pKa.

pHrodo 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. pHrodo iFL Green, pHrodo iFL Red, and pHrodo Deep Red have been combined with 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 or Deep Red 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 SiteClick  pHrodo iFL Red–labeled Trastuzumab (false colored green) and Hoescht 33342 counter stain
High-content analysis of HER2-positive cells incubated with SiteClick 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 amine-reactive dyes and labeling kits

pHrodo iFL products and pHrodo Deep Red are available for traditional antibody labeling of available lysines on antibodies. These amine-reactive pHrodo STP or TFP esters are available in green, red, and deep red versions, as either stand-alone packages or 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