proteinase k (Millipore)
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
![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](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760697/bin/41598_2017_18530_Fig7_HTML.jpg)
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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
![... in its precursor (p) form and as a proteinase K (PK) resistant mature (m) form. COX VIa-L ... 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](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760697/bin/41598_2017_18530_Fig7_HTML.jpg)
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

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

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

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

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

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

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

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:

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:

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

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

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

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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