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Antibody Internalization
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 research applications.
We offer a wide range of antibody labeling technologies and dyes for antibody labeling that can be used to monitor internalization.
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.
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 |  |  |  |
| Photostability | | | |
| Brightness | | | |
| pKa* | 6.5 | 6.5 | 5 |
| Multiplexing | Yes | ||
| Live cells | Yes | ||
| Fixed cells | No | ||
| Platforms | Imaging, high content analysis, flow cytometry | ||
| Zenon Fab Fragment | Z25609 (Mouse) Z25611 (Human) Z25613 (Human 5x) | Z25610 (Mouse) Z25612 (Human) Z25614 (Human 5x) | — |
| SiteClick Site Specific | — | C20034 | S10914 |
| Amine-reactive labeling kit | P36015 (20 μg) P36022 (100 μg) P36023 (1mg) | P36014 (20 μg) P36020 (100 μg) P36021 (1mg) | P35355 (100 μg) P35356 (1mg) |
| 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.
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 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.
Product highlight: pHrodo iFL Red and Green STP Ester dyes
pHrodo iFL Red and Green STP Ester dyes are improved versions of the original pHrodo Red and Green dyes and are optimized to create more soluble conjugates than the original dyes. This enhanced property makes them useful for the labeling of antibodies that may otherwise precipitate out of solution during conjugation.
Antibody labeling for internalization studies
| Name | Labeling scale | SKU |
|---|---|---|
| SiteClick Antibody Azido Modification Kit (sDIBO label is not included order separately) | 100 mg | S20026 |
| Click-iT pHrodo iFL Red sDIBO Alkyne for Antibody Labeling | 100 mg | C20034 |
| SiteClick pHrodo Deep Red sDIBO Alkyne | 250 μg | S10914 |
| SiteClick pHrodo Deep Red sDIBO Alkyne | 1 mg | S10915 |
| SiteClick pHrodo iFL Red sDIBO Alkyne | 1 mg | S10903 |
| SiteClick pHrodo iFL Red sDIBO Alkyne | 5 mg | S10908 |
| Name | Labeling scale | SKU |
|---|---|---|
| Zenon pHrodo iFL Green Mouse IgG Labeling Reagent | Up to four 96 well plates | Z25609 |
| Zenon pHrodo iFL Red Mouse IgG Labeling Reagent | Up to four 96 well plates | Z25610 |
| Zenon pHrodo iFL Green Human IgG Labeling Reagent | Up to four 96 well plates | Z25611 |
| Zenon pHrodo iFL Red Human IgG Labeling Reagent | Up to four 96 well plates | Z25612 |
| Zenon pHrodo iFL Green Human IgG Labeling Reagent 5x | Up to twenty 96 well plates | Z25613 |
| Zenon pHrodo iFL Red Human IgG Labeling Reagent 5x | Up to twenty 96 well plates | Z25614 |
| Name | Labeling scale | SKU |
|---|---|---|
| pHrodo Deep Red Antibody Labeling Kit | 3 x 100 µg | P35355 |
| pHrodo Deep Red Antibody Labeling Kit | 3 x 1 mg | P35356 |
| pHrodo iFL Red Microscale Protein Labeling Kit | 3 x 20 µg | P36014 |
| pHrodo iFL Red Antibody Labeling Kit | 3 x 100 µg | P36020 |
| pHrodo iFL Red Antibody Labeling Kit | 3 x 1 mg | P36021 |
| pHrodo iFL Green Microscale Protein Labeling Kit | 3 x 20 µg | P36015 |
| pHrodo iFL Green Antibody Labeling Kit | 3 x 100 µg | P36022 |
| pHrodo iFL Green Antibody Labeling Kit | 3 x 1 mg | P36023 |
| Name | Labeling scale | SKU |
|---|---|---|
| pHrodo Deep Red TFP Ester (amine-reactive) | 3 x 100 µg | P35358 |
| pHrodo Deep Red TFP Ester (amine-reactive) | 1 mg | P35359 |
| pHrodo iFL Red STP Ester (amine-reactive) | 3 x 100 µg | P36011 |
| pHrodo iFL Red STP Ester (amine-reactive) | 1 mg | P36010 |
| pHrodo iFL Green STP Ester (amine-reactive) | 3 x 100 µg | P36013 |
| pHrodo iFL Green STP Ester (amine-reactive) | 1 mg | P36012 |
| Name | Labeling scale | SKU |
|---|---|---|
| SiteClick Antibody Azido Modification Kit (sDIBO label is not included order separately) | 100 mg | S20026 |
| Click-iT pHrodo iFL Red sDIBO Alkyne for Antibody Labeling | 100 mg | C20034 |
| SiteClick pHrodo Deep Red sDIBO Alkyne | 250 μg | S10914 |
| SiteClick pHrodo Deep Red sDIBO Alkyne | 1 mg | S10915 |
| SiteClick pHrodo iFL Red sDIBO Alkyne | 1 mg | S10903 |
| SiteClick pHrodo iFL Red sDIBO Alkyne | 5 mg | S10908 |
| Name | Labeling scale | SKU |
|---|---|---|
| Zenon pHrodo iFL Green Mouse IgG Labeling Reagent | Up to four 96 well plates | Z25609 |
| Zenon pHrodo iFL Red Mouse IgG Labeling Reagent | Up to four 96 well plates | Z25610 |
| Zenon pHrodo iFL Green Human IgG Labeling Reagent | Up to four 96 well plates | Z25611 |
| Zenon pHrodo iFL Red Human IgG Labeling Reagent | Up to four 96 well plates | Z25612 |
| Zenon pHrodo iFL Green Human IgG Labeling Reagent 5x | Up to twenty 96 well plates | Z25613 |
| Zenon pHrodo iFL Red Human IgG Labeling Reagent 5x | Up to twenty 96 well plates | Z25614 |
| Name | Labeling scale | SKU |
|---|---|---|
| pHrodo Deep Red Antibody Labeling Kit | 3 x 100 µg | P35355 |
| pHrodo Deep Red Antibody Labeling Kit | 3 x 1 mg | P35356 |
| pHrodo iFL Red Microscale Protein Labeling Kit | 3 x 20 µg | P36014 |
| pHrodo iFL Red Antibody Labeling Kit | 3 x 100 µg | P36020 |
| pHrodo iFL Red Antibody Labeling Kit | 3 x 1 mg | P36021 |
| pHrodo iFL Green Microscale Protein Labeling Kit | 3 x 20 µg | P36015 |
| pHrodo iFL Green Antibody Labeling Kit | 3 x 100 µg | P36022 |
| pHrodo iFL Green Antibody Labeling Kit | 3 x 1 mg | P36023 |
| Name | Labeling scale | SKU |
|---|---|---|
| pHrodo Deep Red TFP Ester (amine-reactive) | 3 x 100 µg | P35358 |
| pHrodo Deep Red TFP Ester (amine-reactive) | 1 mg | P35359 |
| pHrodo iFL Red STP Ester (amine-reactive) | 3 x 100 µg | P36011 |
| pHrodo iFL Red STP Ester (amine-reactive) | 1 mg | P36010 |
| pHrodo iFL Green STP Ester (amine-reactive) | 3 x 100 µg | P36013 |
| pHrodo iFL Green STP Ester (amine-reactive) | 1 mg | P36012 |
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