Antibody Internalization | Thermo Fisher Scientific

Streamline your antibody-drug conjugate discovery workflows with our next-gen internalization and degradation assays

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:

Specific late-endosome detection with pHrodo Deep Red
Reproducible, site-specific labeling with SiteClick pHrodo labeling systems
Rapid screening for antibody internalization with Zenon pHrodo reagents
Direct detection of lysosomal degradation using LysoLight Deep Red

Internalization and degradation assay selection guide

Internalization assays
Degradation assays

	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)	Z25622 (Mouse) Z25618 (Human)
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	L36003 (100 µg) L36004 (500 µg)

Internalization assays

	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)	Z25622 (Mouse) Z25618 (Human)
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.

Degradation assays

	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	L36003 (100 µg) L36004 (500 µg)

Internalization assays using pHrodo dye-based assays

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.

Specific late-endosome detection with pHrodo Deep Red

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.

Fluorescent images of SKBR3 cells treated with fluorescent dyes

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.

Site-specifically label with SiteClick pHrodo labeling systems

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.

Figure 2. Overview of SiteClick antibody labeling. The first step in the SiteClick antibody labeling process involves removal of terminal galactose residues from the heavy chain N-linked glycans of the antibody using β-galactosidase, exposing essentially all possible modifiable GlcNAc residues. Second, the free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz to the terminal GlcNAc residues using the GalT(Y289L) enzyme. In the third step, the azide residues are reacted with the dibenzocyclooctyne (DIBO)-functionalized probe of choice (e.g., pHrodo red or deep red sDIBO alkyne). The average degree of labeling is 3–3.5 labels per antibody.

Rapid screening for antibody or ADC internalization with Zenon pHrodo reagents

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.

Figure 3. Internalization study in SKBR3 cells. Five-fold serial dilutions of anti-transferrin receptor were prepared from 10 ng/mL to 0.64 pg/mL while keeping Zenon pHrodo Deep Red Mouse IgG labeling reagent constant at a final assay concentration of 200 nM. After 5-minute labeling, SKBR3 cells were treated with labeled antibodies. Following 16-hour internalization at 37 °C and 5% CO₂ cells were imaged and quantified on the CellInsight CX7 LZR Pro HCS imaging system.

Ordering information for pHrodo dye-based assays

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

SiteClick site specific antibody
labeling kit

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

Zenon domain specific
labeling reagents

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

Amine-reactive
reagents

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

Degradation assays using LysoLight dye-based reagents and kits

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.

Directly monitor lysosomal degradation using LysoLight Deep Red

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.