Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample

Droz, Eric; Taborelli, M.; Wells, Timothy N.C.; Descouts, P.

doi:10.1016/s0006-3495(93)81174-3

Cited by 12 publications

(17 citation statements)

References 25 publications

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“…On the one hand, increased adherence of proteins can be obtained by glow discharge surface treatment, 7,8 chemical modification of the surface with APTES ͑aminopropyltriethoxysilane͒, 9 or ions. On the one hand, increased adherence of proteins can be obtained by glow discharge surface treatment, 7,8 chemical modification of the surface with APTES ͑aminopropyltriethoxysilane͒, 9 or ions.…”

Section: Introductionmentioning

confidence: 99%

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Droz¹

1996

Journal of Vacuum Science &Amp; Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena

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Scanning force microscopy offers the possibility of observing protein molecules under liquid environment. The main difficulty in obtaining reproducible images is given by the low adhesion of the molecules to the substrate. Physisorbed molecules are displaced by the scanning tip or are resuspended in the medium. We have therefore performed a covalent immobilization of immunoglobulin G ͑IgG͒ or its monovalent FabЈ fragment on gold surfaces thanks to thiol groups. For this purpose, multiple thiol groups were chemically introduced into the IgG molecule by treatment with Traut's reagent. As an alternative, for a FabЈ fragment, we prepared molecules with a single thiol group located close to the C terminus of the truncated heavy chain. Both immobilization techniques enable us to observe clearly discernable individual molecules in liquid media. The grafting of FabЈ fragments on gold surface open new opportunities to study protein interactions.

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Section: Introductionmentioning

confidence: 99%

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Droz¹

1996

Journal of Vacuum Science &Amp; Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena

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“…Mica is readily available, and when freshly cleaved forms an extremely smooth surface capable of binding DNA and proteins. Because of this, mica is used in the preparation of replicas of specimens for transmission electron microscopy (Portmann and Koller, 1976;Burkardt and Lurz, 1984) and, more recently, in atomic force microscopy (Bustamante et al, 1992;Lyubchenko et al, 1992, Droz et al, 1993. However, mica has the drawback of being a relatively poor conductor of electrons, ruling out its use in STM.…”

Section: Discussionmentioning

confidence: 99%

Photoelectron imaging of viruses and DNA: evaluation of substrates by unidirectional low angle shadowing and photoemission current measurements

Birrell

Habliston

Griffith

1994

Biophysical Journal

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Photoelectron imaging (photoelectron emission microscopy, PEM or PEEM) is a promising high resolution surface-sensitive technique for biophysical studies. At present, image quality is often limited by the underlying substrate. For photoelectron imaging, the substrate must be electrically conductive, low in electron emission, and relatively flat. A number of conductive substrate materials with relatively low electron emission were examined for surface roughness. Low angle, unidirectional shadowing of the specimens followed by photoelectron microscopy was found to be an effective way to test the quality of substrate surfaces. Optimal results were obtained by depositing approximately 0.1 nm of platinum-palladium (80:20) at an angle of 3 degrees. Among potential substrates for photoelectron imaging, silicon and evaporated chromium surfaces were found to be much smoother than evaporated magnesium fluoride, which initially appeared promising because of its very low electron emission. The best images were obtained with a chromium substrate coated with a thin layer of dextran derivatized with spermidine, which facilitated the spreading and adhesion of biomolecules to the surfaces. Making use of this substrate, improved photoelectron images are reported for tobacco mosaic virus particles and DNA-recA complexes.

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“…The removal of these nonvolatile components is essential for SFM imaging. These low molecular weight contaminants hinder the adsorption of the proteins on the surface and also lower the image contrast by an embedding effect (Droz et al, 1993). Once these components were removed by gel filtration the IgG molecules appeared clearly as individual objects on the surface as shown in Fig.…”

Section: Effect Of Protein Purificaonmentioning

confidence: 96%

“…This technique is routinely applied in electron microscopy. We previously used it to stabilize the T4 bacteriophage (Droz et al, 1993), leading to a more homogeneous distnrbution and increased adherence of viruses on the surface without the need for covalent coupling. Another possible approach is to chemically modify the surface properties of the protein.…”

Section: Introductionmentioning

confidence: 99%

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy

Droz

Taborelli

Descouts

et al. 1994

Biophysical Journal

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Scanning force microscopy has been used successfully to produce images of individual protein molecules. However, one of the problems with this approach has been the high mobility of the proteins caused by the interaction between the sample and the scanning tip. To stabilize the proteins we have modified the adsorption properties of immunoglobulin G on graphite and mica surfaces. We have used two approaches: first, we applied glow discharge treatment to the surface to increase the hydrophilicity, favoring adhesion of hydrophilic protein molecules; second, we used the arginine modifying reagent phenylglyoxal to increase the protein hydrophobicity and thus enhance its adherence to hydrophobic surfaces. We used scanning force microscopy to show that the glow discharge treatment favors a more homogeneous distribution and stronger adherence of the protein molecules to the graphite surface. Chemical modification of the immunoglobulin caused increased aggregation of the proteins on the surface but did not improve the adherence to graphite. On mica, clusters of modified immunoglobulins were also observed and their adsorption was reduced. These results underline the importance of the surface hydrophobicity and charge in controlling the distribution of proteins on the surface.

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Preparation of isolated biomolecules for SFM observations: T4 bacteriophage as a test sample

Cited by 12 publications

References 25 publications

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Photoelectron imaging of viruses and DNA: evaluation of substrates by unidirectional low angle shadowing and photoemission current measurements

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy

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