Structured Review

Merck KGaA proteinase k
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 <t>proteinase</t> 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 .
Proteinase K, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) 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

2) 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

Related Articles

Concentration Assay:

Article Title: New Insight into Antimicrobial Compounds from Food and Marine-sourced Carnobacterium Species through Phenotype and Genome Analyses
Article Snippet: After 5 min of centrifugation at 5000 × g, cell free supernatants (CFSs) were collected and stored at −80 °C. .. To determine whether the active compounds were peptidic, 100 µL CFSs were digested by 1 µL of proteinase K (Proteinase K from Engyodontium album EC 3.4.21.64; Merck, Darmstadt, Germany) at a final concentration of 0.2 mg/mL for 1 h at 37 °C. ..

Lysis:

Article Title: Quantitative and qualitative changes of mitochondria in human preimplantation embryos
Article Snippet: .. Each sample was lysed in 4 μl lysis buffer (20 mM Tris (# 252859, Merck Millipore Co.), 0.4 mg/ml proteinase K (# P2308, Merck Millipore Co.), 0.9% Nonidet P-40 (# 21-3277-2, Merck Millipore Co.), and 0.9% Tween 20 (# P1379, Merck Millipore Co.)) at 55 °C for 30 min, followed by heating at 95 °C for 5 min; the lysate was diluted in DNase-free water to a final volume of 40 μl. .. A 4-μl aliquot of the lysate was added to 12.5 μl Quantifact SYBR Green master mix (Qiagen, Venlo, Limburg, the Netherlands), 8.5 μl DNase-free water and 1 μM of each primer (Table ).

Article Title: Promoter methylation of tumor suppressor genes induced by human papillomavirus in cervical cancer
Article Snippet: Isolation of DNA SiHa, C33a and C33a cells transfected with E6 or E7 were subjected to DNA extraction. .. Briefly, cells were digested with lysis buffer II containing SDS (Sigma-Aldrich; Merck KGaA) and proteinase K (USB) at 50°C overnight. .. Phenol/chloroform extraction and ethanol precipitation were then carried out as previously described ( ).

Incubation:

Article Title: Synthetic Pesticides Used in Agricultural Production Promote Genetic Instability and Metabolic Variability in Candida spp.
Article Snippet: Cell Cycle Phase Determination Fluorescence-activated cell sorting (FACS) analyses were done using an Amnis® FlowSight® flow cytometer and IDEAS software version 6.2.187.0 (Merck Millipore, Warsaw, Poland). .. Briefly, fixed cells in 70% ethanol were incubated with RNase A (1:1 in TE buffer, 1 h, 37 °C) and then digested with proteinase K (1: 1) with Syber Green (1 µL stock solution in DMSO per 1 mL buffer) overnight at 4 °C. ..

Isolation:

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.

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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: 86/100, based on 1 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

    Import of the radiolabeled proteins MTS-Dendra2 and Su9-DHFR into the isolated yeast mitochondria. 35 S-labeled proteins were generated by in vitro transcription/translation in reticulocyte lysate and stored at -80° C. The import reactions were performed essentially as described ( Becker et al., 2009 ). The radiolabeled proteins were thawed and added to the intact and energized mitochondria isolated from the yeast cells. The import reactions were performed at 30° C for the indicated times. In control reactions (-Δψ), the mitochondrial inner membrane potential was depleted. The successful import reactions are characterized by processing of the added precursor (p) form to the mature (m) protein and its resistance against digestion by externally added proteinase K (PK). Both processing and protease resistance do not occur in the absence of a membrane potential. The Su9-DHFR was used as a positive control showing import to the mitochondria.

    Journal: bioRxiv

    Article Title: Accessing mitochondrial protein import in living cells by protein microinjection

    doi: 10.1101/2020.09.30.317412

    Figure Lengend Snippet: Import of the radiolabeled proteins MTS-Dendra2 and Su9-DHFR into the isolated yeast mitochondria. 35 S-labeled proteins were generated by in vitro transcription/translation in reticulocyte lysate and stored at -80° C. The import reactions were performed essentially as described ( Becker et al., 2009 ). The radiolabeled proteins were thawed and added to the intact and energized mitochondria isolated from the yeast cells. The import reactions were performed at 30° C for the indicated times. In control reactions (-Δψ), the mitochondrial inner membrane potential was depleted. The successful import reactions are characterized by processing of the added precursor (p) form to the mature (m) protein and its resistance against digestion by externally added proteinase K (PK). Both processing and protease resistance do not occur in the absence of a membrane potential. The Su9-DHFR was used as a positive control showing import to the mitochondria.

    Article Snippet: The radiolabeled preproteins were added to the isolated energized mitochondria (25 µg total mitochondrial protein per lane) and incubated for up to 30 min at 30° C. To assess complete translocation, the import reactions were divided, and one half was treated with 50 µg/ml proteinase K (Merck, Darmstadt, Germany) to remove all non-imported preproteins.

    Techniques: Isolation, Labeling, Generated, In Vitro, Positive Control

    Import of the radiolabeled proteins into the mitochondria  in vitro Import of the [ 35 S]-labeled protein Su9-DHFR was performed as described in the “Methods” section. The cells (A) or the isolated mitochondria (B) were pretreated before import with PUR (20 µg/ml) or CHX (100 µg/ml) for 30 min at 37°C. The import reactions were incubated for the indicated times. (C) The cells were permeabilized with digitonin after pretreatment, and then import was performed directly without isolation of the mitochondria. In the control reactions (-Δψ), the inner membrane potential was depleted, as described. After import, the cells were treated with proteinase K, as indicated. The imported proteins were analyzed by SDS-PAGE and autoradiography (p: precursor form, m: mature form of Su9-DHFR). The schematic outlines of the experimental procedures are given.

    Journal: bioRxiv

    Article Title: Accessing mitochondrial protein import in living cells by protein microinjection

    doi: 10.1101/2020.09.30.317412

    Figure Lengend Snippet: Import of the radiolabeled proteins into the mitochondria in vitro Import of the [ 35 S]-labeled protein Su9-DHFR was performed as described in the “Methods” section. The cells (A) or the isolated mitochondria (B) were pretreated before import with PUR (20 µg/ml) or CHX (100 µg/ml) for 30 min at 37°C. The import reactions were incubated for the indicated times. (C) The cells were permeabilized with digitonin after pretreatment, and then import was performed directly without isolation of the mitochondria. In the control reactions (-Δψ), the inner membrane potential was depleted, as described. After import, the cells were treated with proteinase K, as indicated. The imported proteins were analyzed by SDS-PAGE and autoradiography (p: precursor form, m: mature form of Su9-DHFR). The schematic outlines of the experimental procedures are given.

    Article Snippet: The radiolabeled preproteins were added to the isolated energized mitochondria (25 µg total mitochondrial protein per lane) and incubated for up to 30 min at 30° C. To assess complete translocation, the import reactions were divided, and one half was treated with 50 µg/ml proteinase K (Merck, Darmstadt, Germany) to remove all non-imported preproteins.

    Techniques: In Vitro, Labeling, Isolation, Incubation, SDS Page, Autoradiography

    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