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Antibody Internalization Assay Using a pH Sensor Fluorescent Dye

  • How a new pH sensor fluorescent dye (pHAb dye) is used to identify antibodies suitable for receptor mediated internalization. 


Therapeutic antibodies conjugated to a toxic small molecule are increasingly being developed.  A key requirement for these antibody drug conjugates (ADCs) is the ability of the antibody to bind to the antigens, typically receptors, on tumor cells and become internalized.  Binding the antigen does not necessarily mean the antibody will become internalized and appears to be epitope specific. Hence, methods that allow selection of antibodies based on ability to be internalized is an important assay early in the hybridoma screening stage.

In this webinar, you will learn about a fluorescent dye that:

  • Fluoresces brightly at low pH but is non-fluorescent at neutral pH
  • Conjugates easily to candidate antibodies with amine- or thiol-reactive chemistry either in colution or while immobilized on protein A or protein G beads
  • Produces a high signal-to-background ratio suitable for plate-based assays as well as fluorescent microsopy and flow cytometry analysis

The utility of the pH sensitive dye will be demonstrated with examples of internalization assays using antibodies like Transtuzumab, Cetuximab and Panitumumab.



Nidhi Nath, PhD
Group Leader, Protein and Nucleic Acid Purification
Promega Corporation

Dr. Nath is a Group Leader in Protein and Nucleic Acid Purification group at Promega Corporation leading development of new tools for antibody and protein purification and functional analysis. Dr. Nath has a Ph.D in Biomedical Engineering from Indian Institute of Technology, Delhi, India.

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