Internalisation and Intracellular Trafficking
Abzena utilises a range of labelling methods to successfully quantify internalisation, or track intracellular trafficking of antibodies, proteins and drug conjugates.
Abzena’s tool box of labelling methods comprises:
- Covalent labelling of proteins with fluorophores using commercially available kits (e.g. NHS chemistry)
- Non-covalent fluorescent labelling of antibody prior cell incubation (e.g. Zenon® labelling technology)
- Detection of internalised proteins in permeabilised cells using secondary detection antibodies
- Preparation of drug conjugates using fluorophores as mock payload (e.g. fluorescent ThioBridge™ reagent)
Internalisation can then be visualised and quantified either using flow cytometry or fluorescence microscopy.
Higher resolution images can also be generated for intracellular trafficking investigation and co-localisation of internalised proteins and intracellular compartments.
Case study: Trastuzumab internalisation
Internalisation of Zenon® labelled Trastuzumab by Her2-expressing SK-BR-3 cells. Her2 receptors on the cell surface were saturated with non-covalently labelled antibody before incubation at 37 °C. After 0 or 24-hour incubation, the membrane-associated signal was either visualised or quenched. Using image analysis, the signal attributed to internalised Trastuzumab was measured and compared to the total fluorescence intensity corresponding to both membrane-bound ant internalised Trastuzumab (non-quenched sample). Right: Higher resolution image of Trastuzumab internalised by Her2-expressing SK-BR-3 cells after 24-hour incubation. Trastuzumab (fuchsia) localised to intracellular vesicles, some of which were identified as lysosomes (green) using an anti-Lamp-1 antibody. Cell nuclei were stained blue.
Working with Abzena
Abzena undertakes bioassays on behalf of clients as standalone projects or as part of larger development projects. We focus on developing the correct bioassay to help you understand your molecule.
To get more information, a quote or to schedule a teleconference please contact us.