Conjugation of fluorescent dyes to Nanobodies

Posted by Dr. Astrid Sitte on Jan 27, 2021 2:01:35 PM

Kévin Dorgt Pexels

The conjugation of the binding domains of camelid heavy chain antibodies, also known as VHHs or Nanobodies, to fluorescent dyes is generating versatile tools for fluorescence microscopy applications like immunofluorescence (IF) and super resolution microscopy (SRM): The small size Nanobodies provide better penetration into tissue and dense cell compartments, plus the small label to epitope displacement provides higher resolution. In addition, the Nanobodies’ high affinity binding to epitope makes them attractive probes for other assays like ELISA.

ChromoTek, part of Proteintech group, provides multiple fluorescent dye (Alexa Fluor® and ATTO) conjugated Nanobodies that are optimized by application:

Nano-Boosters bind to Green and Red Fluorescent proteins (GFP and RFP) and retain and enhance (“boost”) their signals. Nano-Labels are used for staining of non-fluorescent peptide tag or endogenous proteins like the Spot-Tag, histone, and vimentin. Nano-Secondaries bind to primary mouse, rabbit, and human antibodies in a species and subclass specific manner. Though ChromoTek offers a comprehensive set of fluorophores, customers may want to use Nanobodies conjugated to alternative fluorophores that are not available in our catalogue. In order to assist, find below labelling protocols for ChromoTek Nanobodies sorted by labelling chemistry, i.e., NHS and maleimide conjugation.

GFP-Booster and RFP-Booster





Immunostaining with GFP-Booster Alexa Fluor® 488 (green) and RFP-Booster Alexa Fluor® 568 (red). HeLa cells were transiently transfected with Tom70-eGFP and PCNA-mRFP. Scale bar, 10 µm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.


Comparison of labelling chemistries

When planning the conjugation, please consider (i) that the labelling chemistry for conjugation depends on the Nanobody and (ii) the implications of using different labelling chemistries.

Nanobodies with free cysteine(s) (not canonical cysteines!) can be conjugated with maleimide derivatives of dyes, whereas Nanobodies without cysteine(s) can be labelled via NHS derivatives of dyes. Some of ChromoTek’s Nanobodies have sortase tags and can be labelled enzymatically using sortase and corresponding peptide derivatives of dyes.

The conjugation of dyes to cysteine residues provides site-directed labelling with defined degree of labelling (DOL). Conjugated Nanobodies are suitable for quantitation assays. The DOL depends on the quantity of accessible cysteine residues; generally, our Nanobodies have 1 or 2 cysteines.

The conjugation of fluorophores to lysine residues is a rather random labelling and the DOL depends on the equivalents (molar ratio) of dye used. It is recommended to aim for a DOL 1-2 to keep the conjugated Nanobody stable and soluble. This DOL generally works for quantification assays, too. ChromoTek’s Nanobodies without free cysteines are buffered in TAPS, hence they are ready to use. However, the removal of the preservative is recommended.

Finally, the enzymatic conjugation with e.g. sortase requires a sortase tag on both the C- or N-terminus of the Nanobody and on the dye (=fluorophore labelled peptide). The enzymatic labelling is a steady state reaction and you may want to push the reaction towards the products for effective labelling.


Application Notes:


Please contact for more details.



ChromoTek, Spot-Label and Nano-Secondary are registered trademarks of ChromoTek GmbH, part of Proteintech Group. Nanobody is a registered trademark of Ablynx, a Sanofi company. Alexa Fluor is a registered trademark of Life Technologies Corporation, a part of Thermo Fisher Scientific Inc. Other suppliers’ products may be trademarks or registered trademarks of the corresponding supplier each. Statements on other suppliers’ products are given according to our best knowledge.

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