Structured Review

Merck KGaA proteinase k
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.
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Images

1) Product Images from "Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS"

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

Journal: Emerging Microbes & Infections

doi: 10.1038/emi.2017.53

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.
Figure Legend 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.

Techniques Used: Incubation, Western Blot, Fractionation

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.
Figure Legend 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.

Techniques Used: Purification, Western Blot

2) Product Images from "AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp. paracasei BGNJ1-64, Functions in Collagen Adhesion, and Pathogen Exclusion In Vitro"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0126387

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.
Figure Legend 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.

Techniques Used: Confocal Microscopy, Variant Assay, Staining

3) Product Images from "WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization"

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

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02293

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.
Figure Legend 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.

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

4) Product Images from "Structure and Specific Activity of Macrophage-Stimulating Lipopeptides from Mycoplasma hyorhinis"

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

Journal: Infection and Immunity

doi:

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.
Figure Legend 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.

Techniques Used: High Performance Liquid Chromatography, Release Assay

5) Product Images from "De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography"

Article Title: De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography

Journal: IUCrJ

doi: 10.1107/S2052252518009223

Cartoon plot of proteinase K with the anomalous difference density map calculated from all 64 665 indexed derivative images and the phases from the final refined model contoured at 5.0σ. The two Hg atoms (silver spheres) covalently bind to Cys73. The two green spheres correspond to the two bound calcium ions.
Figure Legend Snippet: Cartoon plot of proteinase K with the anomalous difference density map calculated from all 64 665 indexed derivative images and the phases from the final refined model contoured at 5.0σ. The two Hg atoms (silver spheres) covalently bind to Cys73. The two green spheres correspond to the two bound calcium ions.

Techniques Used:

6) Product Images from "An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells"

Article Title: An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells

Journal: Analytical biochemistry

doi: 10.1016/j.ab.2015.10.014

Effects in the ELISA of DNase I, RNase A, and proteinase K treatment. A 25-pg sample of purified PAR was added to each well. (A) Purified PAR and the indicated amount of DNA were incubated with buffer, 1 μg of DNase I, or 1 μg of DNase I with 5 mM MgCl 2 . (B) Purified PAR and the indicated amount of RNA were incubated with buffer or 1 μg RNase A. (C) Purified PAR and the indicated amount of protein were incubated with buffer or 10 μg of proteinase K. The results are the means ± standard deviations from three independent experiments. The data were analyzed for statistical significance using Dunnett’s t -test. *P
Figure Legend Snippet: Effects in the ELISA of DNase I, RNase A, and proteinase K treatment. A 25-pg sample of purified PAR was added to each well. (A) Purified PAR and the indicated amount of DNA were incubated with buffer, 1 μg of DNase I, or 1 μg of DNase I with 5 mM MgCl 2 . (B) Purified PAR and the indicated amount of RNA were incubated with buffer or 1 μg RNase A. (C) Purified PAR and the indicated amount of protein were incubated with buffer or 10 μg of proteinase K. The results are the means ± standard deviations from three independent experiments. The data were analyzed for statistical significance using Dunnett’s t -test. *P

Techniques Used: Enzyme-linked Immunosorbent Assay, Purification, Incubation

7) Product Images from "Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate"

Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.02568

Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
Figure Legend Snippet: Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested with Sac I (lanes 3 and 4), and Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3, Sac I-treated pELF1 excised from PFGE gel; 4, Sac I-treated pELF1 excised from SDS-PFGE gel; 5, Sma I-treated pELF1 excised from PFGE gel; 6, Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.

Techniques Used: Marker

PFGE of the restriction fragments of pELF1 with proteinase K treatment. Lanes: Low Range PFG Marker; proteinase K-treatment only; proteinase K-treated Sal I digests; proteinase K-treated Sma I digests; proteinase K-treated Eag I digests. N. D., not digested.
Figure Legend Snippet: PFGE of the restriction fragments of pELF1 with proteinase K treatment. Lanes: Low Range PFG Marker; proteinase K-treatment only; proteinase K-treated Sal I digests; proteinase K-treated Sma I digests; proteinase K-treated Eag I digests. N. D., not digested.

Techniques Used: Marker

PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 without proteinase K treatment; AA708 with proteinase K treatment; AA708 with both proteinase K and S1 nuclease treatment.
Figure Legend Snippet: PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 without proteinase K treatment; AA708 with proteinase K treatment; AA708 with both proteinase K and S1 nuclease treatment.

Techniques Used: Marker

SDS-PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 with proteinase K treatment; AA708 without proteinase K treatment; AA708 with proteinase K and S1 nuclease treatment.
Figure Legend Snippet: SDS-PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 with proteinase K treatment; AA708 without proteinase K treatment; AA708 with proteinase K and S1 nuclease treatment.

Techniques Used: Marker

8) Product Images from "WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization"

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

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02293

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.
Figure Legend 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.

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

9) Product Images from "Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major"

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major

Journal: Glycobiology

doi: 10.1093/glycob/cwq045

Phosphoglycosylation of reporter secreted acid phosphatase and LPG in the ugp − mutant. ( A ) SAP was expressed in wild type and ugp − mutant, immunoprecipitated with mAb LT8.2 and subjected to western blot analysis with mAb WIC 79.3 (top panel). Loading was checked using mAb LT8.2 (lower panel). Untransfected wild-type cells served as negative control. ( B ) Cell extracts of wild type, ugp − and ugp − / UGP parasites were analyzed by western blotting with mAb WIC79.3. ( C ) Cell extracts of wild type, ugp − and ugp − / UGP parasites were digested with proteinase K and analyzed by western blotting with the Stains-all dye. ( D ) LPG expression of wild type, ugp − and ugp − / UGP parasites was analyzed by indirect immunofluorescence microscopy. Promastigotes were fixed, permeabilized and stained with mAb WIC79.3
Figure Legend Snippet: Phosphoglycosylation of reporter secreted acid phosphatase and LPG in the ugp − mutant. ( A ) SAP was expressed in wild type and ugp − mutant, immunoprecipitated with mAb LT8.2 and subjected to western blot analysis with mAb WIC 79.3 (top panel). Loading was checked using mAb LT8.2 (lower panel). Untransfected wild-type cells served as negative control. ( B ) Cell extracts of wild type, ugp − and ugp − / UGP parasites were analyzed by western blotting with mAb WIC79.3. ( C ) Cell extracts of wild type, ugp − and ugp − / UGP parasites were digested with proteinase K and analyzed by western blotting with the Stains-all dye. ( D ) LPG expression of wild type, ugp − and ugp − / UGP parasites was analyzed by indirect immunofluorescence microscopy. Promastigotes were fixed, permeabilized and stained with mAb WIC79.3

Techniques Used: Mutagenesis, Immunoprecipitation, Western Blot, Negative Control, Expressing, Immunofluorescence, Microscopy, Staining

10) Product Images from "RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues"

Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues

Journal: PLoS ONE

doi: 10.1371/journal.pone.0167336

Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).
Figure Legend Snippet: Schematic workflow for glycosaminoglycan (GAG) extraction including RNase treatment Cell/tissue lysates are treated overnight with proteinase K (Prot. K), followed by DNase-I and RNase-I treatment and finally chloroform extraction and dialysis to remove contaminating proteins/DNA/RNA. After drying/concentration of GAG extracts, the purity of the preparations is assessed using ethidium bromide (EtBr) agarose gel electrophoresis, or by measuring the absorbance at 260 nm (A260).

Techniques Used: Concentration Assay, Agarose Gel Electrophoresis

11) Product Images from "Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria"

Article Title: Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.70.9.5426-5433.2004

(a to f) Effect of permeabilization on hybridization signals in sediment pretreated with 0.1% DEPC. Each triple panel depicts antisense probe staining (a to c) and control probe staining (d to f). (a and d) Nonpermeabilized. (b and e) Permeabilized with lysozyme. (c and f) Permeabilized with lysozyme followed by proteinase K digestion. Bar, 10 μm. (g to j) Effect of permeabilization on hybridization signals in cross sections of B. azoricus gills. pmoA mRNA hybridization after treatment with different proteinase K concentrations is shown. Each double panel depicts hybridization with an antisense probe (g and i) and a control probe (h and j). (g and h) Proteinase K at 0.3 μg ml −1 . (i and j) Proteinase K at 7 μg ml −1 . Bar, 20 μm. (k) Triple hybridization of B. azoricus gill sections in a composite of three images: red, methanotrophic symbionts hybridized with 16S rRNA-targeted, Cy5-labeled probe Baz_meth_845I; blue, thiotrophic symbionts hybridized with 16S rRNA-targeted, Cy3-labeled probe Baz_thio_193; green, pmoA mRNA antisense probe. Bar, 10 μm. (l) Double hybridization of sediment microbes in a composite of two images: red, 16S rRNA hybridized with probe MTMC701-HRP for the detection of Methylomonas , Methylobacter , Methylococcus , and Methylomicrobium ; green, pmoA mRNA. Images were taken with a confocal laser scanning microscope and represent a reconstruction of 21 optical z slices. Bar, 10 μm. (Inset) Reconstruction of four optical z slices. The arrow indicates cells with only a 16S rRNA FISH signal; the arrowheads show cells with only a pmoA mRNA FISH signal; the asterisk indicates cells with both FISH signals.
Figure Legend Snippet: (a to f) Effect of permeabilization on hybridization signals in sediment pretreated with 0.1% DEPC. Each triple panel depicts antisense probe staining (a to c) and control probe staining (d to f). (a and d) Nonpermeabilized. (b and e) Permeabilized with lysozyme. (c and f) Permeabilized with lysozyme followed by proteinase K digestion. Bar, 10 μm. (g to j) Effect of permeabilization on hybridization signals in cross sections of B. azoricus gills. pmoA mRNA hybridization after treatment with different proteinase K concentrations is shown. Each double panel depicts hybridization with an antisense probe (g and i) and a control probe (h and j). (g and h) Proteinase K at 0.3 μg ml −1 . (i and j) Proteinase K at 7 μg ml −1 . Bar, 20 μm. (k) Triple hybridization of B. azoricus gill sections in a composite of three images: red, methanotrophic symbionts hybridized with 16S rRNA-targeted, Cy5-labeled probe Baz_meth_845I; blue, thiotrophic symbionts hybridized with 16S rRNA-targeted, Cy3-labeled probe Baz_thio_193; green, pmoA mRNA antisense probe. Bar, 10 μm. (l) Double hybridization of sediment microbes in a composite of two images: red, 16S rRNA hybridized with probe MTMC701-HRP for the detection of Methylomonas , Methylobacter , Methylococcus , and Methylomicrobium ; green, pmoA mRNA. Images were taken with a confocal laser scanning microscope and represent a reconstruction of 21 optical z slices. Bar, 10 μm. (Inset) Reconstruction of four optical z slices. The arrow indicates cells with only a 16S rRNA FISH signal; the arrowheads show cells with only a pmoA mRNA FISH signal; the asterisk indicates cells with both FISH signals.

Techniques Used: Hybridization, Staining, Labeling, Laser-Scanning Microscopy, Fluorescence In Situ Hybridization

12) Product Images from "De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography"

Article Title: De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography

Journal: IUCrJ

doi: 10.1107/S2052252518009223

Cartoon plot of proteinase K with the anomalous difference density map calculated from all 64 665 indexed derivative images and the phases from the final refined model contoured at 5.0σ. The two Hg atoms (silver spheres) covalently bind to Cys73. The two green spheres correspond to the two bound calcium ions.
Figure Legend Snippet: Cartoon plot of proteinase K with the anomalous difference density map calculated from all 64 665 indexed derivative images and the phases from the final refined model contoured at 5.0σ. The two Hg atoms (silver spheres) covalently bind to Cys73. The two green spheres correspond to the two bound calcium ions.

Techniques Used:

13) Product Images from "Elucidating the Immune Evasion Mechanisms of Borrelia mayonii, the Causative Agent of Lyme Disease"

Article Title: Elucidating the Immune Evasion Mechanisms of Borrelia mayonii, the Causative Agent of Lyme Disease

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02722

Identification and surface exposure of FH/FHL-1-binding proteins in B. mayonii MN14-1420. (A,B) Detection of FH/FHL-1-binding proteins by Far Western blot analysis using NHS as source of FH and purified FHL-1 (750 ng/ml). Cell lysates obtained from B. mayonii MN14-1420, B. burgdorferi LW2, B. burgdorferi B31, B. burgdorferi PKa-1, B. garinii G1, and transformant G1/pCspA_Bmayo were separated by 10% Tris/tricine-SDS-PAGE and transferred onto a nitrocellulose membrane. Flagellin (FlaB) was detected with the monoclonal antibody L41 1C11. The FH-binding proteins (A) were visualized by applying an anti-FH antiserum and FHL-1-binding proteins (B) were detected by using an anti-CCP1-4 antiserum. The corresponding to CspA protein of B. mayonii (Bm) MN14-1420, CspA of B. burgdorferi (Bb) LW2, CspZ, ErpP, and ErpA of B. burgdorferi s.s. are indicated at the right. (C) in situ protease accessibility assay. Native spirochetes were incubated with or without proteinase K or trypsin, then lysed by sonication and total proteins were separated by 10% Tris/tricine-SDS-PAGE. The band corresponding to CspA of B. mayonii is indicated on the right. The mobilities of molecular mass standards are indicated on the left. A full scan of the original membranes is presented in Supplementary Figure 5 .
Figure Legend Snippet: Identification and surface exposure of FH/FHL-1-binding proteins in B. mayonii MN14-1420. (A,B) Detection of FH/FHL-1-binding proteins by Far Western blot analysis using NHS as source of FH and purified FHL-1 (750 ng/ml). Cell lysates obtained from B. mayonii MN14-1420, B. burgdorferi LW2, B. burgdorferi B31, B. burgdorferi PKa-1, B. garinii G1, and transformant G1/pCspA_Bmayo were separated by 10% Tris/tricine-SDS-PAGE and transferred onto a nitrocellulose membrane. Flagellin (FlaB) was detected with the monoclonal antibody L41 1C11. The FH-binding proteins (A) were visualized by applying an anti-FH antiserum and FHL-1-binding proteins (B) were detected by using an anti-CCP1-4 antiserum. The corresponding to CspA protein of B. mayonii (Bm) MN14-1420, CspA of B. burgdorferi (Bb) LW2, CspZ, ErpP, and ErpA of B. burgdorferi s.s. are indicated at the right. (C) in situ protease accessibility assay. Native spirochetes were incubated with or without proteinase K or trypsin, then lysed by sonication and total proteins were separated by 10% Tris/tricine-SDS-PAGE. The band corresponding to CspA of B. mayonii is indicated on the right. The mobilities of molecular mass standards are indicated on the left. A full scan of the original membranes is presented in Supplementary Figure 5 .

Techniques Used: Binding Assay, Far Western Blot, Purification, SDS Page, In Situ, Incubation, Sonication

Related Articles

Centrifugation:

Article Title: WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization
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. .. After 60 min, the precipitate was harvested by centrifugation at 5,000 × g for 15 min at 4°C and resuspended by sonication in 10 ml of distilled water.

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS
Article Snippet: Briefly, late-log phase bacteria (~2 × 109 CFU/mL) were pelleted by centrifugation at 8000g for 10 min at 4 °C and gently resuspended. .. 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.

Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues
Article Snippet: Isolation of GAGs from cells and tissues Original protocol [ , ]: Cell monolayers were thoroughly washed with phosphate-buffered saline (PBS), and digested overnight at 37°C with 125 μg/ml proteinase K (Merck Millipore) in 2 ml extraction buffer (50 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 and 1% triton X-100, pH7.9) per 75 cm2 confluent cell monolayer. .. The digested lysate was then mixed 1:1 with 4 M sodium chloride to dissociate GAG-bound peptides, followed by mixing 1:1 with chloroform and centrifugation for 30 min at 4500xg.

Synthesized:

Article Title: Apoptosis induced by norcantharidin in human tumor cells
Article Snippet: NCTD was synthesized from furan and maleic anhydride via the Diels-Alder reaction. .. RNaseA and proteinase K were purchased from E. Merck, primary antibody (human Bcl-2 specific, rabbit polyclonal antibody) from Santa Cruz Biotechnology, Inc, secondary antibody (biotinylated anti-rabbit IgG) and horseradish peroxidase streptavidin from Vector Laboratories, Inc, and NP-40 and DAB from Sigma.

Incubation:

Article Title: PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast
Article Snippet: .. 50 µl of 20 mg/ml proteinase K (Merck Millipore, 1.24568.0100) were added followed by another hour of incubation at 50°C. ..

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
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. .. Cells were then incubated with 0.03 μmol HI for 15 min at room temperature under constant rotation in the dark.

Article Title: Structure and Specific Activity of Macrophage-Stimulating Lipopeptides from Mycoplasma hyorhinis
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. .. The reaction mixture was separated on an ET 250/4 Nucleosil 120-7 C18 HPLC column (Macherey & Nagel) at 40°C.

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

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS
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. .. After incubation at 37 °C for 1 h, the reaction was stopped by the addition of a 0.5 mM protease inhibitor cocktail (Sigma-Aldrich).

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: For LPG analyses, L. major cell lysates were separated on a 12% gel by SDS–PAGE, transferred onto polyvinylidene difluoride (Millipore) membranes and detected on infrared Li-Cor Odyssey Imager after incubation with WIC79.3 ascites fluid ( ) and goat antimouse IgG IR Dye 800 CW (Li-Cor Biosciences) at dilutions of 1:4000 and 1:20,000, respectively. .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

Western Blot:

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: Paragraph title: Western blot analysis ... The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

High Performance Liquid Chromatography:

Article Title: Structure and Specific Activity of Macrophage-Stimulating Lipopeptides from Mycoplasma hyorhinis
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. .. The reaction mixture was separated on an ET 250/4 Nucleosil 120-7 C18 HPLC column (Macherey & Nagel) at 40°C.

Protease Inhibitor:

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS
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. .. After incubation at 37 °C for 1 h, the reaction was stopped by the addition of a 0.5 mM protease inhibitor cocktail (Sigma-Aldrich).

Confocal Laser Scanning Microscopy:

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
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. .. After washing with PBS, stained cells were added to glass bottom dishes and assayed by confocal laser scanning microscopy using a Leica SP8 system (Leica Microsystems GmbH, Wetzlar, Germany).

Protein Concentration:

Article Title: De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography
Article Snippet: .. Proteinase K was obtained from Merck KGaA and the protein was dissolved to 20 mg ml−1 in protein buffer consisting of 50 mM Tris–HCl pH 7.0, 10 mM CaCl2 , resulting in a final protein concentration of 20 mg ml−1 . ..

Article Title: De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography
Article Snippet: .. Proteinase K was obtained from Merck KGaA and the protein was dissolved to 20 mg ml−1 in protein buffer consisting of 50 m M Tris–HCl pH 7.0, 10 m M CaCl2 , resulting in a final protein concentration of 20 mg ml−1 . ..

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: Protein concentration was measured in triplicate by Bradford protein assay (Biorad, Munich, Germany) to ensure equal loading and checked by reversible Ponceau S staining. .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

Sonication:

Article Title: WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization
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. .. After 60 min, the precipitate was harvested by centrifugation at 5,000 × g for 15 min at 4°C and resuspended by sonication in 10 ml of distilled water.

Pulsed-Field Gel:

Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate
Article Snippet: Paragraph title: Pulsed-Field Gel Electrophoresis (PFGE) ... Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

Isolation:

Article Title: Structure and Specific Activity of Macrophage-Stimulating Lipopeptides from Mycoplasma hyorhinis
Article Snippet: Paragraph title: Isolation of the macrophage-activating lipopeptide MALP-H. ... 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.

Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues
Article Snippet: .. Isolation of GAGs from cells and tissues Original protocol [ , ]: Cell monolayers were thoroughly washed with phosphate-buffered saline (PBS), and digested overnight at 37°C with 125 μg/ml proteinase K (Merck Millipore) in 2 ml extraction buffer (50 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 and 1% triton X-100, pH7.9) per 75 cm2 confluent cell monolayer. .. The lysate was recovered from the culture flask and heated to 95°C for 10 min to deactivate proteinase K before adding 7.5 U/ml DNase-I (Qiagen) and incubating overnight at 37°C.

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: Whole cell lysates from exponentially growing and stationary phase L. major promastigote cultures as well as from amastigotes isolated from mice were separated by SDS–PAGE and transferred onto nitrocellulose membranes (Whatman Schleicher & Schüll, Dassel, Germany). .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

Purification:

Article Title: An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells
Article Snippet: Proteinase K was purchased from Merck Millipore. .. PARantibody(10H, IgG3 kappa)secreted fromhybridoma cellswas purified using a ProteinA SepharoseFast Flowcolumn (GE Healthcare) [ ].

Article Title: Elucidating the Immune Evasion Mechanisms of Borrelia mayonii, the Causative Agent of Lyme Disease
Article Snippet: Purification of FHL-1, FH fragments containing different CCP domains, and the anti-CCP1-4 antiserum has been described previously ( ). .. Proteinase K and trypsin were purchased from Merck (Darmstadt, Germany).

Article Title: RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues
Article Snippet: Isolation of GAGs from cells and tissues Original protocol [ , ]: Cell monolayers were thoroughly washed with phosphate-buffered saline (PBS), and digested overnight at 37°C with 125 μg/ml proteinase K (Merck Millipore) in 2 ml extraction buffer (50 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 and 1% triton X-100, pH7.9) per 75 cm2 confluent cell monolayer. .. The top (aqueous) layer containing purified GAGs was collected and dialyzed thoroughly against 18.2 MΩ.cm deionized water (MQ).

Bradford Protein Assay:

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: Protein concentration was measured in triplicate by Bradford protein assay (Biorad, Munich, Germany) to ensure equal loading and checked by reversible Ponceau S staining. .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

Staining:

Article Title: PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast
Article Snippet: FACS analysis after pheromone induction Cells from an overnight culture grown in SC medium were diluted to an OD600 of 0.025 (∼2.5×105 cells/ml) and grown to an OD600 of 0.4, followed bystimulation with 10 µg/ml of α-factor for 2.5 h. FACS analysis of the DNA content using propidium iodide staining was done as follows: Cells were fixed in 70% ethanol, centrifuged and resuspended in 1 ml of 50 mM sodium citrate containing 0.25 mg/ml DNAse free RNase A (Roche, 10109169001) and incubated for 1 hour at 50°C. .. 50 µl of 20 mg/ml proteinase K (Merck Millipore, 1.24568.0100) were added followed by another hour of incubation at 50°C.

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
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. .. Cells were then incubated with 0.03 μmol HI for 15 min at room temperature under constant rotation in the dark.

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: In addition, in-gel staining with Stains-all (Sigma) was performed according to . .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

Mouse Assay:

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: Whole cell lysates from exponentially growing and stationary phase L. major promastigote cultures as well as from amastigotes isolated from mice were separated by SDS–PAGE and transferred onto nitrocellulose membranes (Whatman Schleicher & Schüll, Dassel, Germany). .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

SDS Page:

Article Title: Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major
Article Snippet: For LPG analyses, L. major cell lysates were separated on a 12% gel by SDS–PAGE, transferred onto polyvinylidene difluoride (Millipore) membranes and detected on infrared Li-Cor Odyssey Imager after incubation with WIC79.3 ascites fluid ( ) and goat antimouse IgG IR Dye 800 CW (Li-Cor Biosciences) at dilutions of 1:4000 and 1:20,000, respectively. .. The cell lysates were previously digested with a mixture of DNase I (0.5 mg mL− 1 , Roche)/RNase A (1 mg mL− 1 , Qiagen, Hilden, Germany) for 2 h at 37°C and subsequently treated with proteinase K (0.5 mg mL-1 , Merck, Darmstadt, Germany) for 2 h at 55°C.

Software:

Article Title: PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast
Article Snippet: 50 µl of 20 mg/ml proteinase K (Merck Millipore, 1.24568.0100) were added followed by another hour of incubation at 50°C. .. Cells were kept at 4°C overnight prior to FACS analysis using a BD FACS Canto II machine and the FACS Diva v6.1.3 software.

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
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. .. Images were acquired and processed with the Leica LAS AF software.

Negative Control:

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS
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. .. As a negative control, proteolysis buffer alone was added to the cell suspension.

Radio Immunoprecipitation:

Article Title: An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells
Article Snippet: Radioimmunoprecipitation assay (RIPA) buffer, DNase I, and RNase A were purchased from Nacalai Tesque. .. Proteinase K was purchased from Merck Millipore.

Concentration Assay:

Article Title: WadD, a New Brucella Lipopolysaccharide Core Glycosyltransferase Identified by Genomic Search and Phenotypic Characterization
Article Snippet: After that, samples were weighed and pipetted into small polycarbonate cap tubes and then suspended by ultrasounds in 2% SDS–60 mM Tris–HCl buffer (pH 6.8) at a concentration of 0.5 g (wet weight) of bacteria per 10 ml of buffer. .. 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.

Article Title: Outer membrane vesicle-associated lipase FtlA enhances cellular invasion and virulence in Francisella tularensis LVS
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. .. After incubation at 37 °C for 1 h, the reaction was stopped by the addition of a 0.5 mM protease inhibitor cocktail (Sigma-Aldrich).

FACS:

Article Title: PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast
Article Snippet: Paragraph title: FACS analysis after pheromone induction ... 50 µl of 20 mg/ml proteinase K (Merck Millipore, 1.24568.0100) were added followed by another hour of incubation at 50°C.

Fluorescence In Situ Hybridization:

Article Title: Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria
Article Snippet: To allow penetration of detection molecules (probes and antibodies), the tissue was digested with proteinase K (7 μg ml−1 ) (from Tritirachium album ; 30 mAnson U mg−1 ; Merck, Darmstadt, Germany) in 50 mM Tris-HCl-5 mM EDTA (pH 8) (TE) for 15 min at RT. .. Slides then were washed three times in MilliQ water for 1 min each time, dehydrated in 70 and 96% ethanol for 1 min each, air dried, and processed for FISH.

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    Merck KGaA proteinase k
    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.
    Proteinase K, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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