proteinase k  (Millipore)

 
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  • 86
    Name:
    Proteinase K from Tritirachium album
    Description:

    Catalog Number:
    p5568
    Price:
    None
    Applications:
    Product has been used to break down cardiac muscle during histopathology studies. It has also been used during the digestion of HEK-293 cells.
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    Structured Review

    Millipore proteinase k
    The assembly of the nuclear-encoded subunit COX VIa-L into mature complex IV and the OXPHOS supercomplexes is reduced in 143B MCAD knockout mitochondria. COX VIa-L was radiolabeled by in vitro transcription/translation, followed by incubation for 10 and 60 min with isolated 143B control (CON) or 143B MCAD knockout (KO) mitochondria. (A) SDS-PAGE showing COX VIa-L in its precursor (p) form and as a <t>proteinase</t> K (PK) resistant mature (m) form. COX VIa-L is imported efficiently into both CON and MCAD KO mitochondria. (B) BN-PAGE showing the assembly of COX VIa-L into the late-stage intermediate (CIV LSI ) and mature complex IV (CIV m ) [following solubilisation in 1% (v/v) TX-100, left] or the CI/CIII 2 /CIV n supercomplexes [following solubilisation in 1% (w/v) digitonin, right]. The amount of newly-translated COX VIa-L assembled into CIV m and the CI/CIII 2 /CIV n supercomplexes was significantly less in MCAD KO mitochondria compared to CON mitochondria after import times of both 10 and 60 min. (C) Quantitation of assembled COX VIa-L into CIV m (n = 4) and the CI/CIII 2 /CIV n supercomplexes (n = 3). Data is mean ± s.d. **p

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    proteinase k - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function"

    Article Title: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18530-4

    The assembly of the nuclear-encoded subunit COX VIa-L into mature complex IV and the OXPHOS supercomplexes is reduced in 143B MCAD knockout mitochondria. COX VIa-L was radiolabeled by in vitro transcription/translation, followed by incubation for 10 and 60 min with isolated 143B control (CON) or 143B MCAD knockout (KO) mitochondria. (A) SDS-PAGE showing COX VIa-L in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. COX VIa-L is imported efficiently into both CON and MCAD KO mitochondria. (B) BN-PAGE showing the assembly of COX VIa-L into the late-stage intermediate (CIV LSI ) and mature complex IV (CIV m ) [following solubilisation in 1% (v/v) TX-100, left] or the CI/CIII 2 /CIV n supercomplexes [following solubilisation in 1% (w/v) digitonin, right]. The amount of newly-translated COX VIa-L assembled into CIV m and the CI/CIII 2 /CIV n supercomplexes was significantly less in MCAD KO mitochondria compared to CON mitochondria after import times of both 10 and 60 min. (C) Quantitation of assembled COX VIa-L into CIV m (n = 4) and the CI/CIII 2 /CIV n supercomplexes (n = 3). Data is mean ± s.d. **p
    Figure Legend Snippet: The assembly of the nuclear-encoded subunit COX VIa-L into mature complex IV and the OXPHOS supercomplexes is reduced in 143B MCAD knockout mitochondria. COX VIa-L was radiolabeled by in vitro transcription/translation, followed by incubation for 10 and 60 min with isolated 143B control (CON) or 143B MCAD knockout (KO) mitochondria. (A) SDS-PAGE showing COX VIa-L in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. COX VIa-L is imported efficiently into both CON and MCAD KO mitochondria. (B) BN-PAGE showing the assembly of COX VIa-L into the late-stage intermediate (CIV LSI ) and mature complex IV (CIV m ) [following solubilisation in 1% (v/v) TX-100, left] or the CI/CIII 2 /CIV n supercomplexes [following solubilisation in 1% (w/v) digitonin, right]. The amount of newly-translated COX VIa-L assembled into CIV m and the CI/CIII 2 /CIV n supercomplexes was significantly less in MCAD KO mitochondria compared to CON mitochondria after import times of both 10 and 60 min. (C) Quantitation of assembled COX VIa-L into CIV m (n = 4) and the CI/CIII 2 /CIV n supercomplexes (n = 3). Data is mean ± s.d. **p

    Techniques Used: Knock-Out, In Vitro, Incubation, Isolation, SDS Page, Polyacrylamide Gel Electrophoresis, Quantitation Assay

    High molecular weight complexes containing MCAD are detectable by BN-PAGE. (A ) MCAD was radiolabeled by in vitro transcription/translation, followed by incubation for 5, 10, 30 and 60 min with isolated 143B control mitochondria. Mitochondria were solubilised in 1% (v/v) TX-100 or 1% (w/v) digitonin, followed by BN-PAGE analysis. MCAD can be detected in complexes of ~175 kDa (MCAD homotetramer) and ~450 kDa (mitochondrial Hsp60 complex), as well as unknown complexes of ~500 kDa (marked *) and ~1,500 kDa (marked #). The intensities of both the ~500 kDa (*) and ~1,500 kDa (#) MCAD-containing complexes (relative to the ~450 kDa mitochondrial Hsp60 complex) was reduced over the 60 min import time course (p = 0.016 and 0.005 respectively). (B) SDS-PAGE showing MCAD in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. MCAD is imported in a mitochondrial membrane potential (Δψ m ) dependent manner, with the levels of mature MCAD protein (m) similar at each time point for both the TX-100 and DIG experiments. (C) BN-PAGE and Western blot analysis of HepG2 cell mitochondria solubilised in 1% (w/v) digitonin (DIG), 0.2% (w/v) dodecyl maltoside (DDM) or 1% (v/v) TX-100. Anti-NDUFA9 antibodies detect monomeric complex I and the CI/CIII 2 /CIV n and CI/CIII 2 OXPHOS supercomplexes, while anti-MCAD antibodies detect MCAD in complexes of ~130 kDa, ~175 kDa (homotetramer), ~450 kDa (mitochondrial Hsp60 complex) and ~1,000 kDa (*).
    Figure Legend Snippet: High molecular weight complexes containing MCAD are detectable by BN-PAGE. (A ) MCAD was radiolabeled by in vitro transcription/translation, followed by incubation for 5, 10, 30 and 60 min with isolated 143B control mitochondria. Mitochondria were solubilised in 1% (v/v) TX-100 or 1% (w/v) digitonin, followed by BN-PAGE analysis. MCAD can be detected in complexes of ~175 kDa (MCAD homotetramer) and ~450 kDa (mitochondrial Hsp60 complex), as well as unknown complexes of ~500 kDa (marked *) and ~1,500 kDa (marked #). The intensities of both the ~500 kDa (*) and ~1,500 kDa (#) MCAD-containing complexes (relative to the ~450 kDa mitochondrial Hsp60 complex) was reduced over the 60 min import time course (p = 0.016 and 0.005 respectively). (B) SDS-PAGE showing MCAD in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. MCAD is imported in a mitochondrial membrane potential (Δψ m ) dependent manner, with the levels of mature MCAD protein (m) similar at each time point for both the TX-100 and DIG experiments. (C) BN-PAGE and Western blot analysis of HepG2 cell mitochondria solubilised in 1% (w/v) digitonin (DIG), 0.2% (w/v) dodecyl maltoside (DDM) or 1% (v/v) TX-100. Anti-NDUFA9 antibodies detect monomeric complex I and the CI/CIII 2 /CIV n and CI/CIII 2 OXPHOS supercomplexes, while anti-MCAD antibodies detect MCAD in complexes of ~130 kDa, ~175 kDa (homotetramer), ~450 kDa (mitochondrial Hsp60 complex) and ~1,000 kDa (*).

    Techniques Used: Molecular Weight, Polyacrylamide Gel Electrophoresis, In Vitro, Incubation, Isolation, SDS Page, Western Blot

    The nuclear-encoded subunit NDUFA9 is imported and assembled efficiently into OXPHOS complex I in 143B MCAD knockout mitochondria. NDUFA9 was radiolabeled by in vitro transcription/translation, followed by incubation for 10 and 60 min with isolated 143B control (CON) or 143B MCAD knockout (KO) mitochondria. (A) SDS-PAGE showing NDUFA9 in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. NDUFA9 is imported efficiently into both CON and MCAD KO mitochondria in a mitochondrial membrane potential (Δψ m ) dependent manner. (B) BN-PAGE showing the assembly of NDUFA9 into the CI/CIII 2 and CI/CIII 2 /CIV supercomplexes (following solubilisation in digitonin, left) or mature complex I (CI, following solubilisation in TX-100, right). (C) Quantitation of NDUFA9 assembly after 60 min of import. There was no difference in the amount of NDUFA9 assembled into the CI/CIII 2 /CIV supercomplex (p = 0.07) or complex I (p = 0.14).
    Figure Legend Snippet: The nuclear-encoded subunit NDUFA9 is imported and assembled efficiently into OXPHOS complex I in 143B MCAD knockout mitochondria. NDUFA9 was radiolabeled by in vitro transcription/translation, followed by incubation for 10 and 60 min with isolated 143B control (CON) or 143B MCAD knockout (KO) mitochondria. (A) SDS-PAGE showing NDUFA9 in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. NDUFA9 is imported efficiently into both CON and MCAD KO mitochondria in a mitochondrial membrane potential (Δψ m ) dependent manner. (B) BN-PAGE showing the assembly of NDUFA9 into the CI/CIII 2 and CI/CIII 2 /CIV supercomplexes (following solubilisation in digitonin, left) or mature complex I (CI, following solubilisation in TX-100, right). (C) Quantitation of NDUFA9 assembly after 60 min of import. There was no difference in the amount of NDUFA9 assembled into the CI/CIII 2 /CIV supercomplex (p = 0.07) or complex I (p = 0.14).

    Techniques Used: Knock-Out, In Vitro, Incubation, Isolation, SDS Page, Polyacrylamide Gel Electrophoresis, Quantitation Assay

    2) Product Images from "Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300"

    Article Title: Enzymes Catalyzing the TCA- and Urea Cycle Influence the Matrix Composition of Biofilms Formed by Methicillin-Resistant Staphylococcus aureus USA300

    Journal: Microorganisms

    doi: 10.3390/microorganisms6040113

    Enzymatic treatment of preformed 24 (no flow) or 17 (flow) h-old biofilms of UAS391-Ery S , ATCC ® 25923™ and urea or TCA-cycle Tn mutants with 100 µg/mL proteinase K ( A , C ) or 100 U/mL DNase I ( B , D ) under no flow (no flow) ( A , B ) and dynamic (flow) ( C , D ) conditions. Blanks refer to incubation in either culture medium with 10 mM Tris-HCl for proteinase K treatment or culture medium for DNase I treatment. Error bars represent 95% confidence intervals. ** refers to p
    Figure Legend Snippet: Enzymatic treatment of preformed 24 (no flow) or 17 (flow) h-old biofilms of UAS391-Ery S , ATCC ® 25923™ and urea or TCA-cycle Tn mutants with 100 µg/mL proteinase K ( A , C ) or 100 U/mL DNase I ( B , D ) under no flow (no flow) ( A , B ) and dynamic (flow) ( C , D ) conditions. Blanks refer to incubation in either culture medium with 10 mM Tris-HCl for proteinase K treatment or culture medium for DNase I treatment. Error bars represent 95% confidence intervals. ** refers to p

    Techniques Used: Flow Cytometry, Incubation

    3) Product Images from "Phenalen-1-one-Mediated Antimicrobial Photodynamic Therapy: Antimicrobial Efficacy in a Periodontal Biofilm Model and Flow Cytometric Evaluation of Cytoplasmic Membrane Damage"

    Article Title: Phenalen-1-one-Mediated Antimicrobial Photodynamic Therapy: Antimicrobial Efficacy in a Periodontal Biofilm Model and Flow Cytometric Evaluation of Cytoplasmic Membrane Damage

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00688

    Spectroscopic measurements for release of nucleic acids. OD medians, 1st and 3rd quartiles of the supernatants of biofilms treated with phenalen-1-one mediated aPDT (groups: PS-L–, PS-L+, SAPYR+L-, SAPYR+L+, SAGUA+L-, and SAGUA+L+) or positive control (lysozyme treatment followed by Proteinase K digestion), as measured at 260 nm for release of nucleic acids.
    Figure Legend Snippet: Spectroscopic measurements for release of nucleic acids. OD medians, 1st and 3rd quartiles of the supernatants of biofilms treated with phenalen-1-one mediated aPDT (groups: PS-L–, PS-L+, SAPYR+L-, SAPYR+L+, SAGUA+L-, and SAGUA+L+) or positive control (lysozyme treatment followed by Proteinase K digestion), as measured at 260 nm for release of nucleic acids.

    Techniques Used: Positive Control

    4) Product Images from "Triple-Helical DNA in Drosophila Heterochromatin"

    Article Title: Triple-Helical DNA in Drosophila Heterochromatin

    Journal: Cells

    doi: 10.3390/cells7120227

    Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.
    Figure Legend Snippet: Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Techniques Used: Staining

    5) Product Images from "Characterization of Antibacterial Cell-Free Supernatant from Oral Care Probiotic Weissella cibaria, CMU"

    Article Title: Characterization of Antibacterial Cell-Free Supernatant from Oral Care Probiotic Weissella cibaria, CMU

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23081984

    Dose-dependent effects of organic acid, hydrogen peroxide (H 2 O 2 ), and a bacteriocin-like compound (BLC) in the cell-free supernatants (CFS) of oraCMU against periodontopathogenic bacteria. Optical density at 600 nm (OD 600 ) of the cell suspension was measured. ( a ) Untreated CFS effect; ( b ) organic acid-dependent effect was measured using proteinase K and catalase-treated CFS; ( c ) H 2 O 2 -dependent effect was measured by the neutralized and proteinase K-treated CFS; ( d ) BLC-dependent effect was evaluated by the neutralized and catalase-treated CFS. P. gingivalis (●); Fusobacterium nucleatum (○); P. intermedia (▼).
    Figure Legend Snippet: Dose-dependent effects of organic acid, hydrogen peroxide (H 2 O 2 ), and a bacteriocin-like compound (BLC) in the cell-free supernatants (CFS) of oraCMU against periodontopathogenic bacteria. Optical density at 600 nm (OD 600 ) of the cell suspension was measured. ( a ) Untreated CFS effect; ( b ) organic acid-dependent effect was measured using proteinase K and catalase-treated CFS; ( c ) H 2 O 2 -dependent effect was measured by the neutralized and proteinase K-treated CFS; ( d ) BLC-dependent effect was evaluated by the neutralized and catalase-treated CFS. P. gingivalis (●); Fusobacterium nucleatum (○); P. intermedia (▼).

    Techniques Used:

    6) Product Images from "Clovamide, a Hydroxycinnamic Acid Amide, Is a Resistance Factor Against Phytophthora spp. in Theobroma cacao"

    Article Title: Clovamide, a Hydroxycinnamic Acid Amide, Is a Resistance Factor Against Phytophthora spp. in Theobroma cacao

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2020.617520

    Enzyme inhibition by clovamide and effect of cacao stage C leaf protein pectinase activity.  (A)  Proteolysis inhibiton by clovamide. “PK” = proteinase K. PK included in all treatments shown, with the addition of ‘Sca6’ protein, clovamide (2 mM), or both. Data represents two experiments ( n  = 5 from each).  (B)  Pectolysis inhibition by clovamide. “Pase” = pectinase from  A. niger . “EGCG” = epigallocatechin gallate. Pase included in all treatments, with the addition of clovamide (2 mM), EGCG (2 mM), ‘Sca6’ stage C leaf protein, or ‘Sca6’ protein in combination with either phenolic compound. Data represents two experiments ( n  = 3 from each).  (C)  Enhancement of pectinase ( A. niger ) activity by cacao stage C leaf protein ( n  = 3). No pectinase (“-Pase”) and pectinase (“+Pase”) with or without addition of ‘ICS1’ or ‘Sca6’ leaf protein. Shared letters mean no difference by Tukey-HSD at  p
    Figure Legend Snippet: Enzyme inhibition by clovamide and effect of cacao stage C leaf protein pectinase activity. (A) Proteolysis inhibiton by clovamide. “PK” = proteinase K. PK included in all treatments shown, with the addition of ‘Sca6’ protein, clovamide (2 mM), or both. Data represents two experiments ( n = 5 from each). (B) Pectolysis inhibition by clovamide. “Pase” = pectinase from A. niger . “EGCG” = epigallocatechin gallate. Pase included in all treatments, with the addition of clovamide (2 mM), EGCG (2 mM), ‘Sca6’ stage C leaf protein, or ‘Sca6’ protein in combination with either phenolic compound. Data represents two experiments ( n = 3 from each). (C) Enhancement of pectinase ( A. niger ) activity by cacao stage C leaf protein ( n = 3). No pectinase (“-Pase”) and pectinase (“+Pase”) with or without addition of ‘ICS1’ or ‘Sca6’ leaf protein. Shared letters mean no difference by Tukey-HSD at p

    Techniques Used: Enzyme Inhibition Assay, Activity Assay, Inhibition

    7) Product Images from "Ferric Reduction Is a Potential Iron Acquisition Mechanism for Histoplasma capsulatum"

    Article Title: Ferric Reduction Is a Potential Iron Acquisition Mechanism for Histoplasma capsulatum

    Journal: Infection and Immunity

    doi:

    Fe(III)-reducing activities of high- and low-MW supernatant components after treatment with proteinase K. Ferric reduction was assayed with BPDS as the chromogenic Fe(II) chelator. The averages of triplicate wells from a representative experiment are shown; standard deviations are indicated by bars. Similar results were obtained in three independent experiments.
    Figure Legend Snippet: Fe(III)-reducing activities of high- and low-MW supernatant components after treatment with proteinase K. Ferric reduction was assayed with BPDS as the chromogenic Fe(II) chelator. The averages of triplicate wells from a representative experiment are shown; standard deviations are indicated by bars. Similar results were obtained in three independent experiments.

    Techniques Used:

    Effects of electron donors on ferric reductase activities of high-MW supernatants. (A) Concentraitons of 5 μM FAD, 5 μM FMN, 50 μM NADH, 50 μM NADPH, and 1.63 mM (0.5 mg/ml) GSH were evaluated for the capacity to increase ferric reductase activity. (B) The role of GSH as a specific electron donor for an enzymatic ferric reductase was examined. GSH was added to a high-MW supernatant that was left untreated, boiled for 15 min, or digested with proteinase K. The untreated high-MW supernatant was supplemented with 0.82 mM (0.5 mg/ml) oxidized glutathione (GSSG). Ferric reduction was assayed with Ferrozine as the chromogenic Fe(II) chelator. The averages of triplicate wells from a representative experiment are shown; standard deviations are indicated by bars. Similar results were obtained in three independent experiments.
    Figure Legend Snippet: Effects of electron donors on ferric reductase activities of high-MW supernatants. (A) Concentraitons of 5 μM FAD, 5 μM FMN, 50 μM NADH, 50 μM NADPH, and 1.63 mM (0.5 mg/ml) GSH were evaluated for the capacity to increase ferric reductase activity. (B) The role of GSH as a specific electron donor for an enzymatic ferric reductase was examined. GSH was added to a high-MW supernatant that was left untreated, boiled for 15 min, or digested with proteinase K. The untreated high-MW supernatant was supplemented with 0.82 mM (0.5 mg/ml) oxidized glutathione (GSSG). Ferric reduction was assayed with Ferrozine as the chromogenic Fe(II) chelator. The averages of triplicate wells from a representative experiment are shown; standard deviations are indicated by bars. Similar results were obtained in three independent experiments.

    Techniques Used: Activity Assay

    8) Product Images from "Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana"

    Article Title: Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057283

    PICL and PICC N-termini face the cytoplasm. Immunoblot analysis using GFP antibody. Microsomal preparations were treated with and without Proteinase K. GFP-CXN and CXN-PAGFP were used as controls. In the microsome fraction containing GFP-CXN, GFP is protected from proteinase K treatment, whereas GFP of CXN-PAGFP is susceptible to proteinase K digestion. GFP of GFP-TDF PICL and GFP-TDF PICC are hydrolyzed indicating exposure to Proteinase K. At the given concentration of proteinase K (sufficient to completely hydrolyze GFP in GFP-TDF PICL and GFP-TDF PICC ), a small amount of PAGFP remains undigested (second column of CXN-PAGFP). Microsomal membranes were solubilized by the detergent Triton X-100. Numbers on the left indicate approximate molecular mass in kilodaltons.
    Figure Legend Snippet: PICL and PICC N-termini face the cytoplasm. Immunoblot analysis using GFP antibody. Microsomal preparations were treated with and without Proteinase K. GFP-CXN and CXN-PAGFP were used as controls. In the microsome fraction containing GFP-CXN, GFP is protected from proteinase K treatment, whereas GFP of CXN-PAGFP is susceptible to proteinase K digestion. GFP of GFP-TDF PICL and GFP-TDF PICC are hydrolyzed indicating exposure to Proteinase K. At the given concentration of proteinase K (sufficient to completely hydrolyze GFP in GFP-TDF PICL and GFP-TDF PICC ), a small amount of PAGFP remains undigested (second column of CXN-PAGFP). Microsomal membranes were solubilized by the detergent Triton X-100. Numbers on the left indicate approximate molecular mass in kilodaltons.

    Techniques Used: Concentration Assay

    9) Product Images from "Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylori *"

    Article Title: Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylori *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.496430

    Purification and DNA binding activity of Tfs4 VirD2. A , DNA co-fractionating in the MBP-VirD2 size exclusion chromatography fraction ( lane 1 ) is efficiently removed in a subsequent ion exchange step ( lane 2 ). B , three-stage purification of MBP-VirD2. *, purified MBP-Tfs4 VirD2 ( lane 1 ) and MBP-Tfs4 VirD2(N) ( lane 2 ) resolved by 10% SDS-PAGE. C , MBP-VirD2 DNA binding activity. Incubation of MBP-VirD2 with 100 ng of pSB14 (containing cloned RP4 oriT ) ( i ), pRD205 (pGEM-TEasy containing cloned tfs4 virD2 and upstream intergenic sequence) ( ii ), or pRD200 (pGEM-TEasy containing cloned tfs4 virD2 only) ( iii ) results in a decrease in supercoiled plasmid and concomitant increase in both open circle (nicked) forms and loading well-retarded nucleoprotein complexes ( black arrow ) as protein concentration increases. Effects are most pronounced with plasmid pRD205. Notably, linear plasmid is also absent from the pRD205 sample following proteinase K treatment. Lanes 1–6 , 0, 0.05, 0.1, 0.15, 0.3, and 0.5 pmol of MBP-VirD2; lane 7 , 0.5 pmol of MBP-VirD2 treated with proteinase K; lane 8 , linear plasmid generated by restriction enzyme digest. All reactions were performed at 37 °C in the presence of MgCl 2 . OC , open circle (nicked); SC , supercoiled, L , linear.
    Figure Legend Snippet: Purification and DNA binding activity of Tfs4 VirD2. A , DNA co-fractionating in the MBP-VirD2 size exclusion chromatography fraction ( lane 1 ) is efficiently removed in a subsequent ion exchange step ( lane 2 ). B , three-stage purification of MBP-VirD2. *, purified MBP-Tfs4 VirD2 ( lane 1 ) and MBP-Tfs4 VirD2(N) ( lane 2 ) resolved by 10% SDS-PAGE. C , MBP-VirD2 DNA binding activity. Incubation of MBP-VirD2 with 100 ng of pSB14 (containing cloned RP4 oriT ) ( i ), pRD205 (pGEM-TEasy containing cloned tfs4 virD2 and upstream intergenic sequence) ( ii ), or pRD200 (pGEM-TEasy containing cloned tfs4 virD2 only) ( iii ) results in a decrease in supercoiled plasmid and concomitant increase in both open circle (nicked) forms and loading well-retarded nucleoprotein complexes ( black arrow ) as protein concentration increases. Effects are most pronounced with plasmid pRD205. Notably, linear plasmid is also absent from the pRD205 sample following proteinase K treatment. Lanes 1–6 , 0, 0.05, 0.1, 0.15, 0.3, and 0.5 pmol of MBP-VirD2; lane 7 , 0.5 pmol of MBP-VirD2 treated with proteinase K; lane 8 , linear plasmid generated by restriction enzyme digest. All reactions were performed at 37 °C in the presence of MgCl 2 . OC , open circle (nicked); SC , supercoiled, L , linear.

    Techniques Used: Purification, Binding Assay, Activity Assay, Size-exclusion Chromatography, SDS Page, Incubation, Clone Assay, Sequencing, Plasmid Preparation, Protein Concentration, Generated

    Site-specific cleavage of oligonucleotides by Tfs4 VirD2. The indicated 5′ (Tfs4) or 3′ (Tfs4, Tfs4 mutated (mut), and RP4) DIG-labeled 30-mer oligonucleotides were incubated with 5 pmol of Tfs4 MBP-VirD2(N) and then subsequently in the presence or absence of either Proteinase K ( K ) or trypsin ( T ). The resulting oligonucleotide products and nucleoprotein-peptide complexes were resolved in denaturing 20% polyacrylamide gels and analyzed by Southern blotting. MBP-VirD2(N) cleaves the 3′-end of the 5′-DIG-labeled Tfs4 oligonucleotide (putative oriT ATCCTG-containing sequence upstream of virD2 in the tfs4 cluster) ( blot 1 ). The equivalent 3′-DIG-labeled oligonucleotide is retained in the gel well in the presence of MBP-VirD2(N) ( blot 2 ). Following protease treatment, cleaved ATCCTG-containing oligonucleotides demonstrate retarded gel migration due to the attachment of proteolyzed VirD2 peptides (D2 Tryp and D2 ProtK , blots 2 and 4 ). Cleavage and VirD2 peptide attachment to 3′-DIG-labeled oligonucleotides can be effectively abrogated by the addition of a 100-fold excess of competing unlabeled Tfs4 oligonucleotide ( C ) but not by the addition of non-competing random sequence oligonucleotide lacking the ATCCTG sequence ( N ) ( blot 2 ). Cleavage is similarly not observed following incubation of MBP-VirD2(N) with a Tfs4 3′-DIG-labeled oligonucleotide in which the ATCCTG sequence is entirely mutated ( mut ; blot 3 ). All reactions required the presence of MgCl 2 . Full oligonucleotide sequences are listed in Table 1 .
    Figure Legend Snippet: Site-specific cleavage of oligonucleotides by Tfs4 VirD2. The indicated 5′ (Tfs4) or 3′ (Tfs4, Tfs4 mutated (mut), and RP4) DIG-labeled 30-mer oligonucleotides were incubated with 5 pmol of Tfs4 MBP-VirD2(N) and then subsequently in the presence or absence of either Proteinase K ( K ) or trypsin ( T ). The resulting oligonucleotide products and nucleoprotein-peptide complexes were resolved in denaturing 20% polyacrylamide gels and analyzed by Southern blotting. MBP-VirD2(N) cleaves the 3′-end of the 5′-DIG-labeled Tfs4 oligonucleotide (putative oriT ATCCTG-containing sequence upstream of virD2 in the tfs4 cluster) ( blot 1 ). The equivalent 3′-DIG-labeled oligonucleotide is retained in the gel well in the presence of MBP-VirD2(N) ( blot 2 ). Following protease treatment, cleaved ATCCTG-containing oligonucleotides demonstrate retarded gel migration due to the attachment of proteolyzed VirD2 peptides (D2 Tryp and D2 ProtK , blots 2 and 4 ). Cleavage and VirD2 peptide attachment to 3′-DIG-labeled oligonucleotides can be effectively abrogated by the addition of a 100-fold excess of competing unlabeled Tfs4 oligonucleotide ( C ) but not by the addition of non-competing random sequence oligonucleotide lacking the ATCCTG sequence ( N ) ( blot 2 ). Cleavage is similarly not observed following incubation of MBP-VirD2(N) with a Tfs4 3′-DIG-labeled oligonucleotide in which the ATCCTG sequence is entirely mutated ( mut ; blot 3 ). All reactions required the presence of MgCl 2 . Full oligonucleotide sequences are listed in Table 1 .

    Techniques Used: Labeling, Incubation, Southern Blot, Sequencing, Migration

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    Western Blot:

    Article Title: MlaFEDB displays flippase activity to promote phospholipid transport towards the outer membrane of Gram-negative bacteria
    Article Snippet: For detection of NBD-lipid fluorescence Silica TLC plates were left unstained and visualised using an Amersham Imager 680 blot and gel imager (GE Healthcare). .. Western blottingIn order to determine the orientation of MlaFEDB once reconstituted into polar lipid liposomes, MlaFEDB:NBD-PE was subjected to Proteinase K digestion. .. 1 mg of MlaFEDB:NBD-PE was incubated with 0.05 mg/ml Proteinase K (Thermo Fisher) for 10 mins at 4 °C.

    other:

    Article Title: Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST
    Article Snippet: After proteinase K digestion, bacteria were pelleted, washed twice with 1 mL PBS 1X and then resuspended in 1 mL PBS 1X.

    Concentration Assay:

    Article Title: Direct Observation of the Interconversion of Normal and Toxic Forms of ?-Synuclein
    Article Snippet: .. To determine the sensitivity of monomers to proteinase K degradation, we used an equimolecular concentration of AF488- and AF647-labeled A90C αS of 70 μM in Tris 25 mM pH 7.4, 0.1 M NaCl; 2 μl of sample were diluted into 500 μl of buffer and 1 μl of proteinase K solution (Proteinase K from Tritirachium album , Sigma-Aldrich ) was added to a final concentration of 0.01, 0.1, 0.4, 2, and 10 μg/ml (i.e., at protein:proteinase K ratios varying from 1:0.0025 to 1:2.5). .. The reaction sample was then incubated for 20 min at 37°C and the reactions stopped by addition of SDS-PAGE loading buffer and loaded in SDS-PAGE gels.

    Electrophoresis:

    Article Title: A Novel, Reliable and Highly Versatile Method to Evaluate Different Prion Decontamination Procedures
    Article Snippet: To investigate whether addition of detergents could improve the efficiency of prion inactivation, prion-coated Teflon® beads were treated for 1 h with bleach or NaOH solutions, both containing 1% Triton-X-100 (Sigma-Aldrich) and subjected to mild shaking. .. Under the same PMSA conditions (see below), using 1 and 2 beads with reaction times of 1, 2, and 4 h and their respective positive and negative controls, the presence or absence of rec-PrPres propagation was evaluated by proteinase K digestion, electrophoresis and total protein staining. .. Preparation and Purification of Recombinant PrP for the PMSA Substrate The bacterial expression of bank vole I109 recombinant prion protein (rec-VoPrP) was achieved using a pOPIN E expression vector prepared using standard molecular biology procedures, as described previously ( ; ; ).

    Staining:

    Article Title: A Novel, Reliable and Highly Versatile Method to Evaluate Different Prion Decontamination Procedures
    Article Snippet: To investigate whether addition of detergents could improve the efficiency of prion inactivation, prion-coated Teflon® beads were treated for 1 h with bleach or NaOH solutions, both containing 1% Triton-X-100 (Sigma-Aldrich) and subjected to mild shaking. .. Under the same PMSA conditions (see below), using 1 and 2 beads with reaction times of 1, 2, and 4 h and their respective positive and negative controls, the presence or absence of rec-PrPres propagation was evaluated by proteinase K digestion, electrophoresis and total protein staining. .. Preparation and Purification of Recombinant PrP for the PMSA Substrate The bacterial expression of bank vole I109 recombinant prion protein (rec-VoPrP) was achieved using a pOPIN E expression vector prepared using standard molecular biology procedures, as described previously ( ; ; ).

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    Millipore proteinase k
    (a) Overview of the orientation distribution of 100 μ m thaumatin crystals (number of crystals n = 69), (b) 40–50 μ m thaumatin crystals (n = 102), and (c) 30 μ m <t>proteinase</t> K crystals (n = 165) mounted in crystallography chips of matching sizes (100, 50, and 30 μ m features). The dots in transparent grey represent the beam directions relative to the crystal coordinate system in Lambert equal-area projection. Dark grey indicates overlap of two or more dots. The centers of the diagrams correspond to the tetragonal c axes, the outermost circle to directions in the a,b-plane. Each “observed” direction is replicated according to the point group (422) but, of all symmetry-related directions, only those pointing to the upper hemisphere are shown. In principle, three parameters are required to define the orientation of a crystal, but for the present purpose, the two angles shown in Figure 3 are sufficient. The third angle, representing rotation of the crystal around the beam direction, is superfluous as this parameter determines the orientation of the diffraction pattern in the detector plane, without changing the pattern itself. Figure prepared with R . 33
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    (a) Overview of the orientation distribution of 100 μ m thaumatin crystals (number of crystals n = 69), (b) 40–50 μ m thaumatin crystals (n = 102), and (c) 30 μ m proteinase K crystals (n = 165) mounted in crystallography chips of matching sizes (100, 50, and 30 μ m features). The dots in transparent grey represent the beam directions relative to the crystal coordinate system in Lambert equal-area projection. Dark grey indicates overlap of two or more dots. The centers of the diagrams correspond to the tetragonal c axes, the outermost circle to directions in the a,b-plane. Each “observed” direction is replicated according to the point group (422) but, of all symmetry-related directions, only those pointing to the upper hemisphere are shown. In principle, three parameters are required to define the orientation of a crystal, but for the present purpose, the two angles shown in Figure 3 are sufficient. The third angle, representing rotation of the crystal around the beam direction, is superfluous as this parameter determines the orientation of the diffraction pattern in the detector plane, without changing the pattern itself. Figure prepared with R . 33

    Journal: Structural Dynamics

    Article Title: Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

    doi: 10.1063/1.4928706

    Figure Lengend Snippet: (a) Overview of the orientation distribution of 100 μ m thaumatin crystals (number of crystals n = 69), (b) 40–50 μ m thaumatin crystals (n = 102), and (c) 30 μ m proteinase K crystals (n = 165) mounted in crystallography chips of matching sizes (100, 50, and 30 μ m features). The dots in transparent grey represent the beam directions relative to the crystal coordinate system in Lambert equal-area projection. Dark grey indicates overlap of two or more dots. The centers of the diagrams correspond to the tetragonal c axes, the outermost circle to directions in the a,b-plane. Each “observed” direction is replicated according to the point group (422) but, of all symmetry-related directions, only those pointing to the upper hemisphere are shown. In principle, three parameters are required to define the orientation of a crystal, but for the present purpose, the two angles shown in Figure 3 are sufficient. The third angle, representing rotation of the crystal around the beam direction, is superfluous as this parameter determines the orientation of the diffraction pattern in the detector plane, without changing the pattern itself. Figure prepared with R . 33

    Article Snippet: In this initial work, we have used commercially available model systems, thaumatin and proteinase K (Sigma Aldrich).

    Techniques:

    Generation of vectors for cellular targeting of F1 expression. (A) The three F1 DNA derivatives used in the study are depicted. pCI-F1 expresses the intact bacterial coding sequence of the F1 anti-gen, pCI-deF1 expresses the putatively mature F1 devoid of the bacterial signal sequence, and pCI-E3/F1 expresses F1 fused to the E3 envelope protein of SFV carrying an eukaryotic signal peptide. (B and C) Radiolabeled, in vitro expression products of pCI-F1, pCI-deF1, and pCI-E3/F1. Polypeptides were generated in a TNT system in the presence or absence of canine microsomes. The β-lactamase cDNA was used as the control for signal cleavage. Purified bacterial F1 was used as the reference for migration of the mature gene product. Prestained, 14 C-labeled polypeptides (Rainbow; Pharmacia) served as MW markers. In panel C, translocation of the E3/F1 hybrid into microsomes was examined by proteinase K treatment in the presence or absence of Triton X-100.

    Journal: Infection and Immunity

    Article Title: Effective Protective Immunity to Yersinia pestis Infection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen

    doi: 10.1128/IAI.71.1.374-383.2003

    Figure Lengend Snippet: Generation of vectors for cellular targeting of F1 expression. (A) The three F1 DNA derivatives used in the study are depicted. pCI-F1 expresses the intact bacterial coding sequence of the F1 anti-gen, pCI-deF1 expresses the putatively mature F1 devoid of the bacterial signal sequence, and pCI-E3/F1 expresses F1 fused to the E3 envelope protein of SFV carrying an eukaryotic signal peptide. (B and C) Radiolabeled, in vitro expression products of pCI-F1, pCI-deF1, and pCI-E3/F1. Polypeptides were generated in a TNT system in the presence or absence of canine microsomes. The β-lactamase cDNA was used as the control for signal cleavage. Purified bacterial F1 was used as the reference for migration of the mature gene product. Prestained, 14 C-labeled polypeptides (Rainbow; Pharmacia) served as MW markers. In panel C, translocation of the E3/F1 hybrid into microsomes was examined by proteinase K treatment in the presence or absence of Triton X-100.

    Article Snippet: Where indicated, translation products were treated with proteinase K (0.2 mg/ml; Sigma) for 15 min at 4°C in the presence or absence of 1% Triton X-100.

    Techniques: Expressing, Sequencing, In Vitro, Generated, Purification, Migration, Labeling, Translocation Assay

    OMVs are produced in vivo by B. thetaiotaomicron and display BtCepA on their surface. (a) Electron microscopic photograph of OMVs collected from a sterile compartment after their diffusion through a 0.22 μm membrane from a compartment containing a B. thetaiotaomicron culture (see the Materials and methods section). Scale bar, ∼100 nm. (b) BtCepA and BtMinpp activities measured after treatment of OMVs with proteinase K. The relative activity is the ratio of the activity measured after proteinase K treatment compared with the activity measured without treatment. Dark grey bars, no proteinase K pre-treatment; light grey bars, proteinase K pre-treatment. * P

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against β-lactam antibiotics

    doi: 10.1093/jac/dku466

    Figure Lengend Snippet: OMVs are produced in vivo by B. thetaiotaomicron and display BtCepA on their surface. (a) Electron microscopic photograph of OMVs collected from a sterile compartment after their diffusion through a 0.22 μm membrane from a compartment containing a B. thetaiotaomicron culture (see the Materials and methods section). Scale bar, ∼100 nm. (b) BtCepA and BtMinpp activities measured after treatment of OMVs with proteinase K. The relative activity is the ratio of the activity measured after proteinase K treatment compared with the activity measured without treatment. Dark grey bars, no proteinase K pre-treatment; light grey bars, proteinase K pre-treatment. * P

    Article Snippet: Enzyme activity at the surface of OMVs A suspension of 250 μg of vesicles in 0.1 M phosphate/1 mM EDTA buffer (pH 7.0) was incubated for 5 min (for vesicles from B. thetaiotaomicron ) and 1 h (for all other Bacteroides species) at 37°C in the presence of 100 mg/L proteinase K (Sigma-Aldrich).

    Techniques: Produced, In Vivo, Diffusion-based Assay, Activity Assay

    UV cross-linking of COS-7 cell proteins on the VEGF mRNA 5′ UTR. (A) Drawing of the different 32 P-labeled RNA probes, obtained from T7 in vitro transcription and corresponding to the complete or parts of the VEGF 5′ UTR mRNA. Relative positions of the 5′ and 3′ ends of each probe are indicated. (B) UV cross-linking experiments performed with probes A to E. S10 COS-7 cell extracts were incubated with 10 6 cpm of the different probes followed by UV irradiation and treatment with RNases A and ONE (see Materials and Methods). The control (Ct) lane corresponds to proteinase K treatment of sample loaded in the first lane. Size markers are indicated. (C) 32 P-labeled probes corresponding to VEGF probe A (complete 5′ UTR) and to EMCV IRES were cross-linked with proteins extracted from S10 COS-7 cells, and the complex was immunoprecipitated with an anti-PTB antibody. The samples were analyzed before (CL) and after (I) immunoprecipitation. The use of a VEGF or EMCV probe is indicated above the lanes. PTB migration is indicated with an arrow.

    Journal: Molecular and Cellular Biology

    Article Title: Two Independent Internal Ribosome Entry Sites Are Involved in Translation Initiation of Vascular Endothelial Growth Factor mRNA

    doi:

    Figure Lengend Snippet: UV cross-linking of COS-7 cell proteins on the VEGF mRNA 5′ UTR. (A) Drawing of the different 32 P-labeled RNA probes, obtained from T7 in vitro transcription and corresponding to the complete or parts of the VEGF 5′ UTR mRNA. Relative positions of the 5′ and 3′ ends of each probe are indicated. (B) UV cross-linking experiments performed with probes A to E. S10 COS-7 cell extracts were incubated with 10 6 cpm of the different probes followed by UV irradiation and treatment with RNases A and ONE (see Materials and Methods). The control (Ct) lane corresponds to proteinase K treatment of sample loaded in the first lane. Size markers are indicated. (C) 32 P-labeled probes corresponding to VEGF probe A (complete 5′ UTR) and to EMCV IRES were cross-linked with proteins extracted from S10 COS-7 cells, and the complex was immunoprecipitated with an anti-PTB antibody. The samples were analyzed before (CL) and after (I) immunoprecipitation. The use of a VEGF or EMCV probe is indicated above the lanes. PTB migration is indicated with an arrow.

    Article Snippet: The samples were then treated with a mix of RNase ONE (5 U; Promega) and 2.5 μg of RNase A at 37°C for 30 min and, when indicated, with proteinase K (Sigma) at 37°C for 20 min at a final concentration of 1 mg/ml.

    Techniques: Labeling, In Vitro, Incubation, Irradiation, Immunoprecipitation, Migration