proteinase k  (Merck KGaA)

Journal: Emerging Microbes & Infections

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS

doi: 10.1038/emi.2017.53

Figure Lengend Snippet: Subcellular localization of FtlA. ( A ) Protease inaccessibility of FtlA. Intact cells of LVS were first incubated with different concentrations of protease K (pK) with or without Triton X-100 permeabilization. FtlA, IglC (cytoplasmic protein control), and FopA (outer membrane protein control) were detected in the cell lysates by Western blotting. ( B ) Distribution of FtlA in subcellular fractions. Subcellular fractionation was performed with intact cells of LVS. FtlA, IglC and FopA were detected in the whole cell lysate (LVS) or subcellular fractions of LVS by Western blotting. The sizes of the proteins are indicated on the right in kDa.

Article Snippet: Proteinase K (Merck, Darmstadt, Germany) in proteolysis buffer (10 mM Tris–HCl, pH 8.0, 5 mM CaCl2 ) was added to final concentrations of 250–1000 μg/mL, or 0.1% Triton X-100 and proteinase K (to a final concentration of 250 μg/mL) were added.

Techniques: Incubation, Western Blot, Fractionation

Journal: Emerging Microbes & Infections

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS

doi: 10.1038/emi.2017.53

Figure Lengend Snippet: Protease accessibility of OMV-associated FtlA. OMVs purified from F. tularensis LVS supernatant were treated with proteinase K in the presence or absence of 0.02% SDS. FtlA and FopA in the samples were probed by Western blotting. The sizes of the proteins are indicated on the right in kDa.

Article Snippet: Proteinase K (Merck, Darmstadt, Germany) in proteolysis buffer (10 mM Tris–HCl, pH 8.0, 5 mM CaCl2 ) was added to final concentrations of 250–1000 μg/mL, or 0.1% Triton X-100 and proteinase K (to a final concentration of 250 μg/mL) were added.

Techniques: Purification, Western Blot

Journal: PLoS ONE

Article Title: AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp. paracasei BGNJ1-64, Functions in Collagen Adhesion, and Pathogen Exclusion In Vitro

doi: 10.1371/journal.pone.0126387

Figure Lengend Snippet: Images of Lb . paracasei subsp. paracasei BGNJ1-64 obtained by confocal microscopy. Agg +— microscopic analysis of cells of auto-aggregation positive strain Lb . paracasei subsp. paracasei BGNJ1-64; Agg - —cells of the aggregation-deficient variant of this strain, not able to form multicellular structures; Agg + /protK—cells of strain BGNJ1-64 treated with proteinase K with loss of ability to form multicellular structures. Higher magnification images are presented on lower panel. Bacteria were stained with hexidium iodide fluorescent dye (excitation maximum of 480 nm and emission maximum of 625 nm). Scale bars are 25 μm for the upper and 5 μm for the lower panel.

Article Snippet: To characterize the proteinaceous nature of the factor(s) involved in aggregation, bacterial cells were resuspended in PBS containing proteinase K (1 mg/ml) (Merck, Darmstadt, Germany) and incubated for 1 h at 37°C prior staining.

Techniques: Confocal Microscopy, Variant Assay, Staining

Journal: Frontiers in Microbiology

Article Title: WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization

doi: 10.3389/fmicb.2018.02293

Figure Lengend Snippet: Mutation of wadD generates an LPS core defect. Left panel, SDS–PAGE electrophoresis and silver staining of SDS–proteinase K extracts; central panel, Western blot analysis of SDS–proteinase K extracts with a polyclonal serum of a B. melitensis -infected rabbit; right panel, Western blot analysis of SDS–proteinase K extracts with monoclonal anti-core antibody A68/24G12/A08.

Article Snippet: This treatment was followed by digestion with 60 μl of proteinase-K at 2.5 mg/ml in HCl–Tris per ml of sample (Merck KGaA) for 3 h at 55°C, and overnight incubation at 20°C.

Techniques: Mutagenesis, SDS Page, Electrophoresis, Silver Staining, Western Blot, Infection

Journal: Infection and Immunity

Article Title: Structure and Specific Activity of Macrophage-Stimulating Lipopeptides from Mycoplasma hyorhinis

doi:

Figure Lengend Snippet: HPLC of octyl glucoside-extracted MSA from M. hyorhinis . MSA, as determined by the nitric oxide release assay (solid line), was eluted with a 2-propanol gradient (straight dashed line). The UV trace at 256 nm is shown as a fainter dashed line. (A) A sample from an octyl glucoside extract of M. hyorhinis clone VIII-23 containing 5.5 mg of protein with 2 × 10 7 U of MSA was applied to a 10- by 250-mm RP18 reversed-phase column. (B) Material eluting in peak 1 was treated with proteinase K and rechromatographed on a 4- by 250-mm RP18 column.

Article Snippet: A 20-μg portion of proteinase K (Merck, Darmstadt, Germany) was added, and the mixture was incubated for 1.5 h at 37°C, after which time the reaction was stopped by heating for 2 min in a boiling-water bath.

Techniques: High Performance Liquid Chromatography, Release Assay