Covalent Immobilization of Single Proteins on Mica for Molecular Recognition Force Microscopy

Klein, Dionne; Stroh, Cordula M.; Jensenius, Henriette; Es, Maarten van; Kamruzzahan, A. S. M.; Stamouli, Amalia; Gruber, Hermann J.; Oosterkamp, Tjerk H.; Hinterdorfer, Peter

doi:10.1002/cphc.200300844

Cited by 33 publications

(38 citation statements)

References 24 publications

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“…For attachment of B. subtilis spores to a solid support, mica was functionalized either by adsorption of poly-L-lysine or by covalent grafting with ethanolamine (Klein et al, 2003), always followed by deposition of the spores. Neither of the two methods was able to give a dense layer of B. subtilis spores though.…”

Section: Resultsmentioning

confidence: 99%

“…One of the interacting molecules (the 'ligand') has been tethered to the AFM tip, preferably by a flexible polyethylene glycol (PEG) chain Ebner et al, 2007). If the complementary 'receptor' molecule is also present in purified form, then it must be tethered to an ultraflat support in a similar way (Klein et al, 2003). Alternatively, the target molecule may be present on the surface of a living cell or an organelle which must be anchored to some flat support.…”

Section: Introductionmentioning

confidence: 98%

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Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Tang

Krajcikova

Zhu

et al. 2007

J of Molecular Recognition

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Coat assembly in Bacillus subtilis serves as a tractable model for the study of the self-assembly process of biological structures and has a significant potential for use in nano-biotechnological applications. In the present study, the morphology of B. subtilis spores was investigated by magnetically driven dynamic force microscopy (MAC mode atomic force microscopy) under physiological conditions. B. subtilis spores appeared as prolate structures, with a length of 0.6-3 microm and a width of about 0.5-2 microm. The spore surface was mainly covered with bump-like structures with diameters ranging from 8 to 70 nm. Besides topographical explorations, single molecule recognition force spectroscopy (SMRFS) was used to characterize the spore coat protein CotA. This protein was specifically recognized by a polyclonal antibody directed against CotA (anti-CotA), the antibody being covalently tethered to the AFM tip via a polyethylene glycol linker. The unbinding force between CotA and anti-CotA was determined as 55 +/- 2 pN. From the high-binding probability of more than 20% in force-distance cycles it is concluded that CotA locates in the outer surface of B. subtilis spores.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Tang

Krajcikova

Zhu

et al. 2007

J of Molecular Recognition

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“…To ensure that the MBL stays close to the mica surface while imaging in buffer, we bound the protein covalently to the surface via side-chain amine groups of the protein (see Materials and Methods). The average density of anchoring sites of the derivatized mica was ∼ 700 μm − 2 , or one binding site per 40 nm × 40 nm, 39 indicating that any protein is at most bound at two positions.…”

Section: Control Measurements In Buffer Solutionmentioning

confidence: 99%

“…For measurements in liquid, the mica disks were modified according to the amine-bis-succinimidyl method described in Klein et al 39 In brief, freshly cleaved mica disks (as above) were incubated overnight at 75°C with a solution of 0.55 g ethanolamine per milliliter of dimethyl sulfoxide. The solution was kept dry with molecular sieve beads.…”

Section: Sample Preparation For Afmmentioning

confidence: 99%

Mannan-Binding Lectin: Structure, Oligomerization, and Flexibility Studied by Atomic Force Microscopy

Jensenius

Klein

Hecke

et al. 2009

Journal of Molecular Biology

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“…Major tasks for the development of such binding protocols are the realization of defined and suitable coupling chemistry and the adjustment of the surface density of the linkers. Different strategies have been used so far for the specific and site-directed binding of biomolecules to surfaces (Wang et al, 2002;Hinterdorfer, 2002;Klein et al, 2003). Here we report on the attachment of red blood cells (RBCs) to mica surfaces for atomic force microscopy studies.…”

Section: Introductionmentioning

confidence: 99%

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM

Kamruzzahan

Kienberger

Stroh

et al. 2004

Biological Chemistry

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The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tappingmode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.

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Covalent Immobilization of Single Proteins on Mica for Molecular Recognition Force Microscopy

Cited by 33 publications

References 24 publications

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Atomic force microscopy imaging and single molecule recognition force spectroscopy of coat proteins on the surface of Bacillus subtilis spore

Mannan-Binding Lectin: Structure, Oligomerization, and Flexibility Studied by Atomic Force Microscopy

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM

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