What types of antibodies can be labeled with the pHintra™ Antibody Labeling Kit?

The kit uses an amine-based covalent labeling strategy to couple the pH-sensitive dye to primary amine groups on the antibody surface. It works with antibodies from all species and across subclasses, making it suitable for a broad range of experimental scenarios — including antibodies from unconventional species or rare subclasses.

How long does the antibody labeling process take?

The labeling requires only about 30 minutes of hands-on time, and the full experiment is completed in approximately 2.5 hours. After a simple spin-column purification, the labeled antibody can be used directly in internalization assays without additional processing.

What detection methods are compatible with the labeled antibodies?

After labeling and purification, the labeled antibody can be used directly in antibody internalization assays based on both flow cytometry (FACS) and fluorescence cell imaging. The dye (Em660) has an excitation wavelength of 643 nm and an emission wavelength of 660 nm, compatible with standard APC detection channels.

What is the difference between the pHintra™ Antibody Labeling Kit and the pHintra™ Antibody Internalization Detection Reagent?

The Labeling Kit (Cat. No. ALK-A004) uses amine-based covalent labeling, works with antibodies from all species and subclasses, requires about 30 minutes of hands-on time, and is ideal when broad antibody compatibility or labeling stability is needed. The Internalization Detection Reagent (Cat. No. IGG-PZF2001) is a ready-to-use reagent that non-covalently labels the human IgG Fc region within 10 minutes without purification, making it ideal for rapid high-throughput screening of human IgG antibodies and IgG-type ADCs.

Introduction

Q: How does the pH-sensitive dye detect antibody internalization?

The pH-sensitive dye is minimally fluorescent at neutral pH (7.4). Outside the cell, a labeled antibody binds its antigen on the cell surface with low background. Once internalized by endocytosis, the antibody is routed from early endosomes (pH 6.5) through late endosomes (pH 5.0) to lysosomes (pH 4.7). As the vesicle lumen gradually acidifies, the dye is activated and emits bright fluorescence — providing a direct, high signal-to-noise readout that the antibody has completed cellular internalization.

In ADC development and antibody functional research, internalization efficiency and intracellular trafficking are core developability attributes. Labeling requirements also differ from one experimental stage to the next: high-throughput sample screening prioritizes ease of operation, while studies involving antibodies from unconventional species or rare subclasses call for broader compatibility and greater labeling stability.

To support these different internalization assay scenarios, ACROBiosystems has launched the pHintra™ Antibody Labeling Kit. The kit uses an amine-based covalent labeling strategy to efficiently couple a pH-sensitive fluorescent dye to IgG antibodies from multiple species and subclasses. After a simple spin-column purification, the labeled antibody can be used directly in antibody internalization assays based on flow cytometry or cell imaging — a more flexible detection tool for antibody and ADC research.

How It Works

The pHintra™ Antibody Labeling Kit provides a rapid, simple method for covalently attaching a pH-sensitive dye to antibodies through primary amine groups on the antibody surface. The labeled antibody can then be used to measure antibody internalization efficiency by flow cytometry or cell imaging.

The pH-sensitive dye (Em660) emits bright fluorescence in acidic environments while keeping background noise low, with an excitation wavelength of 643 nm and an emission wavelength of 660 nm.

pHintra antibody labeling kit principle for pH-sensitive antibody internalization detection.

Figure: Technical principles of pHintra™ Antibody Labeling Kit

The dye is minimally fluorescent at neutral pH. Outside the cell (pH 7.4), a labeled antibody binds its antigen on the cell surface. Once the antibody is internalized by endocytosis, it is routed from early endosomes (pH 6.5) through late endosomes (pH 5.0) to lysosomes (pH 4.7). As the vesicle lumen gradually acidifies, the dye is activated and emits bright fluorescence — providing a direct readout that the antibody has completed cellular internalization.

Product Information

Cat. No.	Product Description	Order
ALK-A004	pH-Sensitive Fluorescent Dye Antibody Labeling Kit (Em660, 100 μg/Reaction)	Order →

Key Features

Direct labeling, no secondary antibody. The pH-sensitive dye is covalently attached to the target antibody, adding only a small increase in molecular weight and improving assay flexibility.
High signal-to-noise ratio. Strong fluorescence with low background in acidic environments improves the sensitivity of internalization detection.
Acid-responsive fluorescence. The dye brightens only in acidic intracellular compartments such as endosomes and lysosomes, faithfully reporting true antibody internalization.
Just 30 minutes of hands-on time. Antibody labeling requires only about 30 minutes of manual work, and the full experiment is completed in about 2.5 hours.
Broad antibody compatibility. Works with antibodies from all species and across subclasses, fitting more experimental scenarios.
All-in-one reagent set. All required reagents are provided in a single kit with a standardized workflow, eliminating method development.

Labeling Workflow at a Glance

pHintra antibody labeling workflow using pH-sensitive fluorescent dye and spin-column purification.

Figure: Quick guide — antibody labeling workflow

Download the datasheet (DS) for the detailed protocol

Validation Data

Labeled Antibodies Perform in FACS Internalization Assays


Anti-Her2 Ab and Human IgG1 isotype control were labeled using pH-Sensitive Fluorescent Dye Antibody Labeling Kit (Cat. No. ALK-A004). SK-BR-3 cells were treated with Dye Labeled Anti-Her2 Ab and Dye Labeled Isotype Control separately at 37℃ for 4 hours, then analysis by Flow cytometric. APC signal was used to evaluate the internalization signal. Request Protocol	Anti-CD20 Ab and Human IgG1 isotype control were labeled using pH-Sensitive Fluorescent Dye Antibody Labeling Kit (Cat. No. ALK-A004). Raji cells were treated with Dye Labeled Anti-CD20 Ab and Dye Labeled Isotype Control separately at 37℃ for 4 hours, then analysis by Flow cytometric. APC signal was used to evaluate the internalization signal. Request Protocol

Anti-Her2 Ab and Human IgG1 isotype control were labeled using pH-Sensitive Fluorescent Dye Antibody Labeling Kit (Cat. No. ALK-A004). SK-BR-3 cells were treated with Dye Labeled Anti-Her2 Ab and Dye Labeled Isotype Control separately at 37℃ for 4 hours, then analysis by Flow cytometric. APC signal was used to evaluate the internalization signal.

Request Protocol

Anti-CD20 Ab and Human IgG1 isotype control were labeled using pH-Sensitive Fluorescent Dye Antibody Labeling Kit (Cat. No. ALK-A004). Raji cells were treated with Dye Labeled Anti-CD20 Ab and Dye Labeled Isotype Control separately at 37℃ for 4 hours, then analysis by Flow cytometric. APC signal was used to evaluate the internalization signal.

Request Protocol

Labeled Antibodies Perform in Fluorescence Imaging Internalization Assays

Fluorescence imaging of anti-HER2 antibody internalization using pH-sensitive fluorescent labeling.

Application of pH-Sensitive Fluorescent Dye Antibody Labeling Kit in Endocytosis Detection
SK-BR-3 cells were treated with Anti-Her2 Abs-pH-Sensitive Fluorescent Dye Antibody Labeling Kit conjugate and IgG1 Isotype-pH-Sensitive Fluorescent Dye Antibody Labeling Kit conjugate separately for 20 hours (red), then stained with NucBlue Live ReadyProbes (blue) for 20 minutes and imaged on the Cytation 5. A. IgG1 Isotype-pH-Sensitive Fluorescent Dye Antibody Labeling Kit conjugate. B. Anti-Her2 Abs-pH-Sensitive Fluorescent Dye Antibody Labeling Kit conjugate.

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Related Products

Working with a human IgG antibody? Try the ready-to-use internalization detection reagent.

pHintra™ Antibody Internalization Detection Reagent (Cat. No. IGG-PZF2001), featuring a pH-sensitive red fluorescent dye that labels the human IgG Fc region. The reagent forms a stable fluorescent complex within 10 minutes, enabling rapid assessment of antibody internalization processes. View product details →

Choose the Internalization Assay Format That Fits

Category	Ready-to-Use Internalization Assay	Labeling Internalization Assay
Labeling Method	Fc-specific non-covalent labeling	Primary amine-based covalent labeling
Antibody Compatibility	Human IgG antibodies & ADCs	Antibodies/ADCs from different species and subclasses
Preparation Time	Approximately 10 min antibody labeling	30 min hands-on work
Detection Method	Flow cytometry / Cell imaging	Flow cytometry / Cell imaging
Best Suited For	Rapid screening of human IgG antibodies and human IgG-based ADCs	Direct labeling of non-Fc antibodies, antibodies from different species or subclasses, and ADCs
Core Advantage	No purification required, approximately 10 min labeling	Broad compatibility across antibody species and subclasses, as well as ADCs