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Antibody Internalization and Degradation Assays for ADC Discovery |
When antibodies bind to cell-surface receptors, they trigger endocytosis, internalizing the receptors and antibodies into the cell. This process is vital in research of targeted drug delivery to cancer cells, especially with antibody-drug conjugates (ADCs). ADCs are engineered antibodies linked to potent cytotoxic molecules, which can be trafficked to the lysosome for drug release and tumor cell death or recycled through the endosomal pathway.
Understanding the intracellular trafficking pathways is crucial for advancing drug discovery. ADCs or antibodies can be linked to our pH-sensitive pHrodo dyes to monitor internalization or to our LysoLight dyes to confirm lysosomal degradation, offering easy-to-use tools for effective drug discovery.
Explore pHrodo dye– or LysoLight dye–based assay offerings:
pHrodo Green | pHrodo Red | pHrodo Deep Red | |
---|---|---|---|
Organelle | 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/Fixed cells | Live | ||
Applications | Imaging, high content analysis, flow cytometry | ||
SiteClick Site Specific | — | C20034 | S10914 |
Zenon Fab Fragment | Z25609 (Mouse) Z25611 (Human) Z25613 (Human 5x) | Z25610 (Mouse) Z25612 (Human) Z25614 (Human 5x) | |
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.
LysoLight Deep Red | |
---|---|
Range | Lysosome |
Common filter set | Cy5 |
Ex/Em (nm) | 650/668 |
Signal-to-noise ratio | |
Photostability | |
Brightness | |
pKa | N/A |
Multiplexing | Yes |
Live Cells | Yes |
Fixed Cells | No |
Platforms | Imaging, high content analysis, flow cytometry |
Zenon Fab Fragment | N/A |
SiteClick Site Specific | N/A |
Amine-reactive labeling kit | L36001 (100 µg) L36002 (1 mg) |
Amine-reactive reagent |
pHrodo Green | pHrodo Red | pHrodo Deep Red | |
---|---|---|---|
Organelle | 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/Fixed cells | Live | ||
Applications | Imaging, high content analysis, flow cytometry | ||
SiteClick Site Specific | — | C20034 | S10914 |
Zenon Fab Fragment | Z25609 (Mouse) Z25611 (Human) Z25613 (Human 5x) | Z25610 (Mouse) Z25612 (Human) Z25614 (Human 5x) | |
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.
LysoLight Deep Red | |
---|---|
Range | Lysosome |
Common filter set | Cy5 |
Ex/Em (nm) | 650/668 |
Signal-to-noise ratio | |
Photostability | |
Brightness | |
pKa | N/A |
Multiplexing | Yes |
Live Cells | Yes |
Fixed Cells | No |
Platforms | Imaging, high content analysis, flow cytometry |
Zenon Fab Fragment | N/A |
SiteClick Site Specific | N/A |
Amine-reactive labeling kit | L36001 (100 µg) L36002 (1 mg) |
Amine-reactive reagent |
Invitrogen pHrodo kits and reagents utilize pH-sensitive technology to allow researchers to study endocytosis or phagocytosis with high specificity. Available in red, green, and deep red, pHrodo dyes are nonfluorescent at the neutral pH of the extracellular environment. Only upon internalization into the acidic environment of the endosome or lysosome, does the pH-sensitive dye brightly fluoresce. This minimizes background fluorescence and enables detection without the use of quenchers or wash steps in internalization assays.
The pH-sensitive technology of Invitrogen pHrodo dyes can be combined with other labeling technologies. These include the site-specific technology of SiteClick antibody labeling and the rapid Zenon antibody labeling methods, to suit your specific internalization assay needs.
pHrodo Deep Red has a lower pKA than pHrodo red and green, which means it will not fluoresce until later in the endocytic pathway. This delayed fluorescence allows for the monitoring of different stages within the endocytic pathway, providing valuable insights into the internalization process.
Figure 1.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.
The SiteClick pHrodo labeling system utilizes copper-free click chemistry to site-specifically attach pHrodo dyes to antibodies for antibody internalization studies. The site-specific attachment of pHrodo dyes preserves antibody functionality, while minimizing background noise, for more precise research.
The SiteClick Azido Modification Kits attach azido moieties to the carbohydrate domains on the heavy chains of IgG antibodies. This modification allows for specific and reproducible antibody labeling using pHrodo dyes or other labels, using SiteClick sDIBO alkynes. Researchers can then use the resulting conjugate, which enables detailed monitoring of intracellular trafficking, in the development of antibody-drug conjugates.
Invitrogen Zenon pHrodo IgG Labeling reagents harnesses the efficient Zenon antibody labeling technology with pH-sensitive pHrodo dye technology for rapid screening of antibody internalization. Zenon is a non-covalent labeling method that employs Fab fragments coupled to pHrodo or Alexa Fluor dyes. These Fab fragments bind to the Fc portion of human or mouse IgG antibody to form a labeling complex in under 5 minutes.
Zenon pHrodo labeled Fab fragments provide a bright fluorescence-based indication of antibody uptake and trafficking in target cells. As the pH becomes bore acidic, the pHrodo dyes increase in fluorescence. This makes Zenon pHrodo labeling reagents excellent for measuring endocytic activity based on the acidification of labeled antibodies ingested by the cell.
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 |
Zenon pHrodo iFL Deep Red Mouse IgG Labeling reagent | Up to four 96 well plates | Z25622 |
Zenon pHrodo iFL Deep Red Human IgG Labeling reagent | Up to four 96 well plates | Z25618 |
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 |
---|---|---|
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 |
Zenon pHrodo iFL Deep Red Mouse IgG Labeling reagent | Up to four 96 well plates | Z25622 |
Zenon pHrodo iFL Deep Red Human IgG Labeling reagent | Up to four 96 well plates | Z25618 |
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 |
Invitrogen LysoLight antibody labeling kits and reactive dyes are powerful tools for monitoring catabolic lysosomal degradation of antibodies, proteins, or antibody-drug conjugates (ADCs). Unlike our pHrodo technology which relies on pH-gradients, LysoLight conjugated antibodies or proteins remain non-fluorescent even in the late endosome. The LysoLight dye uniquely fluoresces once it reaches the protease-rich environment of the lysosome, indicating proteolysis.
This mechanism offers exceptional sensitivity and specificity to understand the internalization and trafficking of a protein or ADC. Using LysoLight technology, researchers can also assess the propensity of targeted protein degrader candidates towards catabolic or recycling pathways. This can help researchers in the targeted protein degradation field fine-tune their candidates for maximum therapeutic potential. LysoLight kits and reagents are compatible with a wide range of platforms such as flow cytometry, high content imaging, and IncuCyte® Live-Cell Analysis Systems.
Read the Mechanisms of Protein Degradation article to learn more.
LysoLight Deep Red is an excellent sensor designed to directly monitor lysosomal degradation of target proteins. In its un-cleaved form, LysoLight Deep Red has no background fluorescence. Only upon cleavage by the lysosomally localized protease, cathepsin B, does the dye become fluorescent, leading to bright fluorescence in the Cy5 channel. The sensor contains an SDP ester allowing it to be conjugated to lysine residues on antibodies or other proteins of interest to monitor protein degradation effectively.
For Research Use Only. Not for use in diagnostic procedures.