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Keysight Technologies
Attaching Antibodies to AFM Probes with the
Sulfhydryl Reactive PEG Tether, NHS-PEG18-PDP




                                 Application Note
Introduction
AFM probes can be transformed into sensitive, chemically selective biosensors by
attaching ligand molecules to the tips of the probes. Single-molecule molecular
recognition force microscopy (MRFM) is an AFM-based technique that relies heavily
on probes that have been modified with ligand molecules [Riener et al. 2003,
Hinterdorfer 2004]. In MRFM, single-molecule unbinding interactions between ligands
and complementary receptors that are immobilized on a substrate are observed and
quantified one by one as the AFM cantilever approaches and subsequently withdraws
from the substrate. These force spectroscopy (FS) experiments can provide valuable
information about the structure and dynamics of molecular unbinding events at the
single-molecule level [Noy et al. 1997]. The technique has also been effectively applied
to gain an understanding of the intramolecular forces involved in protein folding and
polymer elongation [Allison et al. 2002].

Topography and recognition imaging (TREC) is another single-molecule AFM
technique that utilizes ligands on an AFM probe and complementary receptors on
a substrate. TREC resolves recognition maps of ligand-receptor interactions by
scanning an AFM probe, which contains immobilized ligands, over a substrate, which
contains receptors, in magnetic AC (MAC) Mode. Using a Keysight AFM equipped with
PicoTREC, which resolves the TREC AFM signals, the lateral positions of functionally
active receptors can be resolved with nanometer resolution [Stroh et al. 2004a,
Stroh et al. 2004b, Kienberger et al. 2004b]. PicoTREC has been used to image, map,
and analyze the chemical compositions of a variety of samples, including molecular
interactions between nucleic acids and proteins [Lin et al. 2006], antibodies and
antigens [Marcus et al. 2006, Stroh et al. 2004a, Stroh et al. 2004b], and small ligands
and their receptors [Ebner et al. 2005].

There are many biochemical immobilization and bioconjugation chemistry schemes
that have been applied to the investigation of ligand-receptor interactions by MRFM
and TREC imaging. In such studies, biological ligands are typically bound to the tip
of an AFM probe, such as a MAC lever, while corresponding receptor molecules are
bound to a flat substrate, such as mica, silicon, flat glass, or a gold-coated substrate.

This protocol describes the attachment of antibodies to AFM probes via short
polyethylene glycol (PEG) linkers. The heterobifunctional, amine and sulfhydryl
reactive PEG linker, PDP-PEG18-NHS, should be synthesized in an organic chemistry
laboratory [Haselgruber et al. 1995, Kamruzzahan et al. 2006] or purchased from a
vendor that performs custom synthesis. The AFM probes will be cleaned, activated
with an alkoxy aminosilane, PEGylated with PDP-PEG18-NHS, and then conjugated
with the antibody.

The antibodies will be activated with sulfhydryl groups so that they can couple
effectively with the sulfhydryl reactive PEG linker. With minor modifications, the
methods described in this protocol may also be applicable to other sulfhydryl reactive
NHS-PEG linkers, including NHS-PEG12-maleimide and NHS-PEG24-maleimide (both
of which are available from Quanta BioDesign of Powell, Ohio USA) and NHS-PEG27-
maleimide and NHS-PEG29-SS-Pyr (available from PolyPure of Oslo, Norway) as well
as other commercially available or custom-synthesized sulfhydryl reactive NHS-PEGn
linkers. However, it should be noted that longer or shorter linkers may not be as well
suited to TREC imaging as PEG18 [Kamruzzahan et al. 2006].
03 | Keysight | Attaching Antibodies to AFM Probes with the Sulfhydryl Reactive PEG Tether, NHS-PEG18-PDP 

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