Modification of the Polaron sputter‐coater unit for glow‐discharge activation of carbon support films

Benada, Oldřich; Pokorný, Václav

doi:10.1002/jemt.1060160304

Cited by 39 publications

(22 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Each sample was then incubated with the respective serum (diluted 1:20 in 1x TBS) for 1 h. Then, protein A coupled immunogold particles (Protein A - 20 nm colloidal gold, Sigma-Aldrich) was added 1:20 in 1x TBS for 1 h. After immunogold labelling, 10 μL of the phage stock solution was adsorbed on carbon-coated copper grids (Athene 200, Plano, Wetzlar/Germany) that had been glow discharged shortly before use [21]. The suspensions were allowed to adsorb for 5 min, unbound material was removed by touching the grid to filter paper.…”

Section: Methodsmentioning

confidence: 99%

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

Ploss

Kühn

2011

BMC Microbiol

View full text Add to dashboard Cite

BackgroundFilamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles.ResultsThe amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags.ConclusionsOur results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

show abstract

Section: Methodsmentioning

confidence: 99%

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

Ploss

Kühn

2011

BMC Microbiol

View full text Add to dashboard Cite

show abstract

“…5-l drops of protein solutions were applied onto glow dischargeactivated carbon-coated grids (46) and adsorbed for 30 s. The excess of solution was blotted with filter paper, and the grids were immediately negatively stained with 2% uranyl acetate in double-distilled H 2 O for 30 s. The grids were blotted with filter paper and air-dried. The samples were examined in Philips CM100 electron microscope at 80 kV and magnification of 64,000ϫ.…”

Section: Methodsmentioning

confidence: 99%

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Wald

Osičková

Šulc

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Ameloblastin plays a key role in the complex biomineralization process that forms tooth enamel, the hardest tissue of the body. Results: Ameloblastin self-associates into ribbon-like supramolecular structures via a short segment encoded by exon 5. Conclusion: Ameloblastin self-association may be essential for correct structural organization and mineralization of the enamel in vivo. Significance: The results provide molecular insight into biology of tooth enamel formation.

show abstract

“…This step was repeated two times, since sheep erythrocytes do not have the CD11b/CD18 receptor for CyaA and CyaA loses its activity in solution. The erythrocytes were then hypotonicaly lysed with 10 mM HEPES (pH 7.4) and erythrocyte ghosts were deposited onto glow-discharge activated grids coated with formvar-carbon film (Benada and Pokorny, 1990 For scanning electron microscopy, the toxin-treated erythrocytes were fixed with 3% glutaraldehyde, washed with a cacodylate buffer, and allowed to sediment for 48 h at 48C onto SPI-pore filter (0.2 lm) treated with poly-L-lysine (Sanders et al, 1975). The samples were dehydrated in alcohol series followed by absolute acetone, and dried in a critical-point device (Balzers 010).…”

mentioning

confidence: 99%

Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes

Vojtová

Kofroňová

Šebo

et al. 2006

Microscopy Res & Technique

View full text Add to dashboard Cite

Adenylate cyclase toxin (CyaA) of Bordetella pertussis penetrates the membrane of eukaryotic cells, producing high levels of intracellular cAMP, as well as hemolysis that results from the formation of cation-selective toxin channels in the membrane. Using several microscopical approaches we studied the effects of CyaA action on the morphology of sheep erythrocytes during early phases preceding lysis and examined localization of CyaA molecules within the erythrocyte membrane. CyaA induced a cascade of morphological changes of erythrocytes, such as shrinkage, formation of membrane projections, and blebs and swelling. The use of an enzymatically inactive CyaA-AC- toxoid that is unable to produce cAMP and of a CyaA-E581K mutant exhibiting higher hemolytic activity than with CyaA showed that the hemolytic activity is responsible for the induction of morphological changes of erythrocytes. Further, immunolabeling of inserted CyaA-232/FLAG molecules with specific anti-FLAG antibodies and IgG-gold particles indicated a clustered distribution of CyaA molecules in erythrocyte membrane. This was confirmed by immunofluorescence and confocal microscopy, which revealed uniform stoichiometry of CyaA clusters, suggesting CyaA binding into specific domains in erythrocyte membrane. Indeed, a decrease of CyaA binding after cholesterol depletion of erythrocytes suggests toxin targeting and binding to membrane microdomains (rafts).

show abstract

Modification of the Polaron sputter‐coater unit for glow‐discharge activation of carbon support films

Cited by 39 publications

References 6 publications

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes

Contact Info

Product

Resources

About