proteinase k  (Thermo Fisher)


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    Proteinase K
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    Structured Review

    Thermo Fisher proteinase k
    NOCT protein is localized to the mitochondria, dependent on its N-terminal mitochondrial targeting sequence. a) Immunofluorescence analysis of intracellular localization of NOCT constructs. NOCT-3F is localized to the mitochondria whereas NOCT Δ( 2 - 15 )-3F and NOCT Δ( 2 - 67 )-3F constructs, which lack the MTS, are predominantly localized to the cytoplasm. The NOCT expression plasmids indicated on the left were transfected into 143B human osteosarcoma cells and visualized using anti-FLAG monoclonal antibody immunofluorescence against the C-terminal 3x-FLAG epitope on each construct with Alexa Fluor Plus 488 secondary antibody (green). Mitochondria are visualized with MitoTracker Red CMXRos (red) and nuclei are visualized with DAPI (blue). GST-3F served as a control. The white scale bar is 10 µm. b) NOCT fractionates with the mitochondria, as tested by western blotting of subcellular fractions (including total, cytosol, and mitochondria) prepared from HEK293T cells that were transfected with NOCT (1-431)-3F. The mitochondrial fractions were divided into three equal aliquots and were either left untreated or were treated with <t>Proteinase</t> K or Proteinase K with Triton X-100. The fractions were then analyzed by SDS PAGE and western blotting with the indicated antibodies. NOCT was detected using guinea pig polyclonal anti-NOCT and goat monoclonal anti-DDDDK (FLAG) antibodies. Fractionation was validated using the following antibodies: anti-uL4m and anti-uS15m for the mitochondrial matrix, anti-Mitofusin 2 and anti-TOM20 for the outer mitochondrial membrane, anti-Histone H3 for the nucleus, and anti-uL1 for the cytoplasm.

    https://www.bioz.com/result/proteinase k/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide dinucleotide metabolites"

    Article Title: Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide dinucleotide metabolites

    Journal: bioRxiv

    doi: 10.1101/2020.01.12.903534

    NOCT protein is localized to the mitochondria, dependent on its N-terminal mitochondrial targeting sequence. a) Immunofluorescence analysis of intracellular localization of NOCT constructs. NOCT-3F is localized to the mitochondria whereas NOCT Δ( 2 - 15 )-3F and NOCT Δ( 2 - 67 )-3F constructs, which lack the MTS, are predominantly localized to the cytoplasm. The NOCT expression plasmids indicated on the left were transfected into 143B human osteosarcoma cells and visualized using anti-FLAG monoclonal antibody immunofluorescence against the C-terminal 3x-FLAG epitope on each construct with Alexa Fluor Plus 488 secondary antibody (green). Mitochondria are visualized with MitoTracker Red CMXRos (red) and nuclei are visualized with DAPI (blue). GST-3F served as a control. The white scale bar is 10 µm. b) NOCT fractionates with the mitochondria, as tested by western blotting of subcellular fractions (including total, cytosol, and mitochondria) prepared from HEK293T cells that were transfected with NOCT (1-431)-3F. The mitochondrial fractions were divided into three equal aliquots and were either left untreated or were treated with Proteinase K or Proteinase K with Triton X-100. The fractions were then analyzed by SDS PAGE and western blotting with the indicated antibodies. NOCT was detected using guinea pig polyclonal anti-NOCT and goat monoclonal anti-DDDDK (FLAG) antibodies. Fractionation was validated using the following antibodies: anti-uL4m and anti-uS15m for the mitochondrial matrix, anti-Mitofusin 2 and anti-TOM20 for the outer mitochondrial membrane, anti-Histone H3 for the nucleus, and anti-uL1 for the cytoplasm.
    Figure Legend Snippet: NOCT protein is localized to the mitochondria, dependent on its N-terminal mitochondrial targeting sequence. a) Immunofluorescence analysis of intracellular localization of NOCT constructs. NOCT-3F is localized to the mitochondria whereas NOCT Δ( 2 - 15 )-3F and NOCT Δ( 2 - 67 )-3F constructs, which lack the MTS, are predominantly localized to the cytoplasm. The NOCT expression plasmids indicated on the left were transfected into 143B human osteosarcoma cells and visualized using anti-FLAG monoclonal antibody immunofluorescence against the C-terminal 3x-FLAG epitope on each construct with Alexa Fluor Plus 488 secondary antibody (green). Mitochondria are visualized with MitoTracker Red CMXRos (red) and nuclei are visualized with DAPI (blue). GST-3F served as a control. The white scale bar is 10 µm. b) NOCT fractionates with the mitochondria, as tested by western blotting of subcellular fractions (including total, cytosol, and mitochondria) prepared from HEK293T cells that were transfected with NOCT (1-431)-3F. The mitochondrial fractions were divided into three equal aliquots and were either left untreated or were treated with Proteinase K or Proteinase K with Triton X-100. The fractions were then analyzed by SDS PAGE and western blotting with the indicated antibodies. NOCT was detected using guinea pig polyclonal anti-NOCT and goat monoclonal anti-DDDDK (FLAG) antibodies. Fractionation was validated using the following antibodies: anti-uL4m and anti-uS15m for the mitochondrial matrix, anti-Mitofusin 2 and anti-TOM20 for the outer mitochondrial membrane, anti-Histone H3 for the nucleus, and anti-uL1 for the cytoplasm.

    Techniques Used: Sequencing, Immunofluorescence, Construct, Expressing, Transfection, FLAG-tag, Western Blot, SDS Page, Fractionation

    2) Product Images from "dCas9-targeted locus-specific protein isolation method identifies histone gene regulators"

    Article Title: dCas9-targeted locus-specific protein isolation method identifies histone gene regulators

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1718844115

    Layout of the CLASP. Graphic depiction of the CLASP method. (1) Cells of interest are crosslinked with a crosslinker of choice. (2) Small fragments of chromatin are generated by mechanical shearing of the fixed cells. (3) Recombinant dCas9-3×FLAG loaded with the chosen guide RNA is added to the chromatin mixture. (4) Anti-FLAG antibody conjugated to resin is added to RNP/chromatin and washed; enriched chromatin is eluted with 3×FLAG peptide. (5) Through the use of either Proteinase K or nuclease treatment, enriched DNA or protein samples can be isolated for downstream applications.
    Figure Legend Snippet: Layout of the CLASP. Graphic depiction of the CLASP method. (1) Cells of interest are crosslinked with a crosslinker of choice. (2) Small fragments of chromatin are generated by mechanical shearing of the fixed cells. (3) Recombinant dCas9-3×FLAG loaded with the chosen guide RNA is added to the chromatin mixture. (4) Anti-FLAG antibody conjugated to resin is added to RNP/chromatin and washed; enriched chromatin is eluted with 3×FLAG peptide. (5) Through the use of either Proteinase K or nuclease treatment, enriched DNA or protein samples can be isolated for downstream applications.

    Techniques Used: Generated, Recombinant, Isolation

    3) Product Images from "Prion Pathogenesis is Independent of Caspase-12"

    Article Title: Prion Pathogenesis is Independent of Caspase-12

    Journal: Prion

    doi:

    ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.
    Figure Legend Snippet: ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.

    Techniques Used: Infection, Mouse Assay, Western Blot, Migration

    Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.
    Figure Legend Snippet: Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.

    Techniques Used: Mouse Assay, Staining

    Related Articles

    Purification:

    Article Title: Intracellular Infection by the Human Granulocytic Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis
    Article Snippet: Periodate-treated ehrlichiae were prepared by incubating purified HGE agent with 20 mM sodium periodate (Sigma Chemical Co., St. Louis, Mo.) in 50 mM sodium acetate buffer (pH 4.5) for 1 h at room temperature in the dark followed by incubation with 50 mM sodium borohydride (Sigma) in sterile phosphate-buffered saline (PBS; 2.7 mM KCl–1.8 mM KH2 PO4 –137 mM NaCl–10 mM NaH2 PO4 , pH 7.4) for 30 min at room temperature ( , ). .. For proteinase K treatment, the purified HGE agent was incubated in 1 mg of proteinase K (GIBCO)/ml in distilled water at 60°C for 2 h. After incubation, 1 mM phenylmethylsulfonyl fluoride (Sigma) was added, the mixture was incubated for 10 min at 60°C, and then the ehrlichiae were washed three times in RPMI 1640 medium ( , ). .. Neutrophils were isolated from buffy coats from healthy donors (Ohio Red Cross, Columbus, Ohio).

    Incubation:

    Article Title: Intracellular Infection by the Human Granulocytic Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis
    Article Snippet: Periodate-treated ehrlichiae were prepared by incubating purified HGE agent with 20 mM sodium periodate (Sigma Chemical Co., St. Louis, Mo.) in 50 mM sodium acetate buffer (pH 4.5) for 1 h at room temperature in the dark followed by incubation with 50 mM sodium borohydride (Sigma) in sterile phosphate-buffered saline (PBS; 2.7 mM KCl–1.8 mM KH2 PO4 –137 mM NaCl–10 mM NaH2 PO4 , pH 7.4) for 30 min at room temperature ( , ). .. For proteinase K treatment, the purified HGE agent was incubated in 1 mg of proteinase K (GIBCO)/ml in distilled water at 60°C for 2 h. After incubation, 1 mM phenylmethylsulfonyl fluoride (Sigma) was added, the mixture was incubated for 10 min at 60°C, and then the ehrlichiae were washed three times in RPMI 1640 medium ( , ). .. Neutrophils were isolated from buffy coats from healthy donors (Ohio Red Cross, Columbus, Ohio).

    Article Title: Transcriptional Regulation of Multi-Drug Tolerance and Antibiotic-Induced Responses by the Histone-Like Protein Lsr2 in M. tuberculosis
    Article Snippet: .. Briefly, 200 ng of ΦX174 RT DNA (Promega) was incubated in the presence of different units (2–12 units) of topoisomerase I (Invitrogen), with and without Lsr2, and incubated at 37 °C for 30 min. After incubation, 6% SDS and proteinase K (4 mg/ml; Invitrogen) were added to the mixture, which was incubated for 15 min at 37 °C. .. Samples were run on a 0.7% agarose gel and then stained with ethidium bromide (Sigma) for analysis.

    Article Title: Identification of the Xenopus DNA2 protein as a major nuclease for the 5?- > 3? strand-specific processing of DNA ends
    Article Snippet: A typical SSA assay contained 0.5 µl 10× ATP mix, 5 µl xDNA2-depleted (supplemented with the purified xDNA2 or ELB) or mock-depleted NPE, and 20 ng/µl DNA in a 7.5 µl reaction. .. After incubation at room temperature, 1.8 µl samples were taken at the indicated times and mixed with 1.8 µl 2% SDS/25 mM EDTA, 6.4 µl H2 O, and 1 µl Proteinase K. After incubation at room temperature for 6 h, the DNA samples were separated on 1% TAE/agarose gels and detected by SYBR Gold (Invitrogen). .. Antibody preparation and immunodepletion of xDNA2 The cDNA encoding the N-terminal 712 amino acids of xDNA2 was isolated by PCR and subcloned into a pGEX expression vector.

    Article Title: dCas9-targeted locus-specific protein isolation method identifies histone gene regulators
    Article Snippet: .. Twenty units of DnaseI (New England Biolabs) were added to the washed resin in 1× NT2 buffer with 150 mM NaCl and incubated at 37 °C for 30 min. SDS was added to mixture to get a 0.1% final concentration, and the mixture was treated with 2.5 uL of Proteinase K (Thermo Fisher) at 56 °C for 1 h. RNA was isolated by using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols. cDNA synthesis was performed with 50 ug of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad) and was diluted 10-fold. .. Real-time PCR analysis was carried out with SYBR Select Master Mix for CFX (Life Technologies) using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

    Article Title: Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep
    Article Snippet: Up to six rounds of PMCA were performed for each sample. sPMCA end-point was detection of the proteinase K resistant misfolded prion protein core (PrPres ) by western blotting. sPMCA products generated from PBMC of groups 2 and 3 along with the PBMC isolated from known scrapie-infected VRQ/VRQ sheep (4124 and 4125) [ , ] and scrapie-uninfected ARQ/VRQ sheep were analyzed by western blot as previously described [ , ]. .. Briefly, 10 μl of sPMCA products were diluted 1:2 with 2X lysis buffer (20 mM Tris–HCl (pH 7.5), 1 % NP-40, 1 % sodium deoxycholate) prior to incubation with proteinase K (200 μg/mL final concentration) at 37 °C for 90 min. After addition of NuPAGE LDS sample buffer and NuPAGE sample reducing reagent (Invitrogen, Carlsbad, CA) and boiling for 10 min samples (equivalent to 5.5 μl sPMCA product) were loaded onto a 12 % Nu-PAGE Bis-Tris gel (Invitrogen, Carlsbad, CA). .. After electrophoresis, proteins were transferred onto PVDF membranes, blocked with commercial casein blocker (Pierce, Rockford, IL) containing 0.05 % Tween 20.

    Concentration Assay:

    Article Title: dCas9-targeted locus-specific protein isolation method identifies histone gene regulators
    Article Snippet: .. Twenty units of DnaseI (New England Biolabs) were added to the washed resin in 1× NT2 buffer with 150 mM NaCl and incubated at 37 °C for 30 min. SDS was added to mixture to get a 0.1% final concentration, and the mixture was treated with 2.5 uL of Proteinase K (Thermo Fisher) at 56 °C for 1 h. RNA was isolated by using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols. cDNA synthesis was performed with 50 ug of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad) and was diluted 10-fold. .. Real-time PCR analysis was carried out with SYBR Select Master Mix for CFX (Life Technologies) using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

    Article Title: Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep
    Article Snippet: Up to six rounds of PMCA were performed for each sample. sPMCA end-point was detection of the proteinase K resistant misfolded prion protein core (PrPres ) by western blotting. sPMCA products generated from PBMC of groups 2 and 3 along with the PBMC isolated from known scrapie-infected VRQ/VRQ sheep (4124 and 4125) [ , ] and scrapie-uninfected ARQ/VRQ sheep were analyzed by western blot as previously described [ , ]. .. Briefly, 10 μl of sPMCA products were diluted 1:2 with 2X lysis buffer (20 mM Tris–HCl (pH 7.5), 1 % NP-40, 1 % sodium deoxycholate) prior to incubation with proteinase K (200 μg/mL final concentration) at 37 °C for 90 min. After addition of NuPAGE LDS sample buffer and NuPAGE sample reducing reagent (Invitrogen, Carlsbad, CA) and boiling for 10 min samples (equivalent to 5.5 μl sPMCA product) were loaded onto a 12 % Nu-PAGE Bis-Tris gel (Invitrogen, Carlsbad, CA). .. After electrophoresis, proteins were transferred onto PVDF membranes, blocked with commercial casein blocker (Pierce, Rockford, IL) containing 0.05 % Tween 20.

    Isolation:

    Article Title: dCas9-targeted locus-specific protein isolation method identifies histone gene regulators
    Article Snippet: .. Twenty units of DnaseI (New England Biolabs) were added to the washed resin in 1× NT2 buffer with 150 mM NaCl and incubated at 37 °C for 30 min. SDS was added to mixture to get a 0.1% final concentration, and the mixture was treated with 2.5 uL of Proteinase K (Thermo Fisher) at 56 °C for 1 h. RNA was isolated by using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols. cDNA synthesis was performed with 50 ug of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad) and was diluted 10-fold. .. Real-time PCR analysis was carried out with SYBR Select Master Mix for CFX (Life Technologies) using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

    Lysis:

    Article Title: Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep
    Article Snippet: Up to six rounds of PMCA were performed for each sample. sPMCA end-point was detection of the proteinase K resistant misfolded prion protein core (PrPres ) by western blotting. sPMCA products generated from PBMC of groups 2 and 3 along with the PBMC isolated from known scrapie-infected VRQ/VRQ sheep (4124 and 4125) [ , ] and scrapie-uninfected ARQ/VRQ sheep were analyzed by western blot as previously described [ , ]. .. Briefly, 10 μl of sPMCA products were diluted 1:2 with 2X lysis buffer (20 mM Tris–HCl (pH 7.5), 1 % NP-40, 1 % sodium deoxycholate) prior to incubation with proteinase K (200 μg/mL final concentration) at 37 °C for 90 min. After addition of NuPAGE LDS sample buffer and NuPAGE sample reducing reagent (Invitrogen, Carlsbad, CA) and boiling for 10 min samples (equivalent to 5.5 μl sPMCA product) were loaded onto a 12 % Nu-PAGE Bis-Tris gel (Invitrogen, Carlsbad, CA). .. After electrophoresis, proteins were transferred onto PVDF membranes, blocked with commercial casein blocker (Pierce, Rockford, IL) containing 0.05 % Tween 20.

    Article Title: Prion Pathogenesis is Independent of Caspase-12
    Article Snippet: The following antibodies and dilutions were used: 6H4 anti-PrP 1:10,000 (Prionics), SAF83 anti-PrP 1:3000 (Cayman), anti-Caspase-12, 1:5,000 (Exalpha); anti-Grp58, 1:5,000 (StressGene), 1:1,000 anti-phospho JNK and anti-JNK (Cell Signaling Technology). .. For proteinase K treatment, 10% brain homogenates from terminally ill WT or Caspase-12 KOs were diluted to 1% in lysis buffer and treated with 50 µg/ml proteinase K (Invitrogen) for one hour at 37°C. ..

    Activity Assay:

    Article Title: Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide dinucleotide metabolites
    Article Snippet: The protein concentration of the total cellular, cytoplasmic, and mitochondrial fractions was measured using the DC Lowry protein assay (BioRad) and split into 3 equal aliquots. .. Mitochondrial fractions were subjected to no treatment, treatment with Proteinase K (20 μg/4 mg mitochondrial protein, Thermo Fisher), or treatment with Proteinase K + 1% Triton X-100 to expose intramitochondrial proteins to protease activity. .. Equal volumes of treated mitochondrial fractions, and cytoplasmic and nuclear fractions with protein concentrations equal to the untreated mitochondrial fraction were resolved via SDS PAGE followed by Western blotting as described above.

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    Thermo Fisher proteinase k
    NOCT protein is localized to the mitochondria, dependent on its N-terminal mitochondrial targeting sequence. a) Immunofluorescence analysis of intracellular localization of NOCT constructs. NOCT-3F is localized to the mitochondria whereas NOCT Δ( 2 - 15 )-3F and NOCT Δ( 2 - 67 )-3F constructs, which lack the MTS, are predominantly localized to the cytoplasm. The NOCT expression plasmids indicated on the left were transfected into 143B human osteosarcoma cells and visualized using anti-FLAG monoclonal antibody immunofluorescence against the C-terminal 3x-FLAG epitope on each construct with Alexa Fluor Plus 488 secondary antibody (green). Mitochondria are visualized with MitoTracker Red CMXRos (red) and nuclei are visualized with DAPI (blue). GST-3F served as a control. The white scale bar is 10 µm. b) NOCT fractionates with the mitochondria, as tested by western blotting of subcellular fractions (including total, cytosol, and mitochondria) prepared from HEK293T cells that were transfected with NOCT (1-431)-3F. The mitochondrial fractions were divided into three equal aliquots and were either left untreated or were treated with <t>Proteinase</t> K or Proteinase K with Triton X-100. The fractions were then analyzed by SDS PAGE and western blotting with the indicated antibodies. NOCT was detected using guinea pig polyclonal anti-NOCT and goat monoclonal anti-DDDDK (FLAG) antibodies. Fractionation was validated using the following antibodies: anti-uL4m and anti-uS15m for the mitochondrial matrix, anti-Mitofusin 2 and anti-TOM20 for the outer mitochondrial membrane, anti-Histone H3 for the nucleus, and anti-uL1 for the cytoplasm.
    Proteinase K, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

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    NOCT protein is localized to the mitochondria, dependent on its N-terminal mitochondrial targeting sequence. a) Immunofluorescence analysis of intracellular localization of NOCT constructs. NOCT-3F is localized to the mitochondria whereas NOCT Δ( 2 - 15 )-3F and NOCT Δ( 2 - 67 )-3F constructs, which lack the MTS, are predominantly localized to the cytoplasm. The NOCT expression plasmids indicated on the left were transfected into 143B human osteosarcoma cells and visualized using anti-FLAG monoclonal antibody immunofluorescence against the C-terminal 3x-FLAG epitope on each construct with Alexa Fluor Plus 488 secondary antibody (green). Mitochondria are visualized with MitoTracker Red CMXRos (red) and nuclei are visualized with DAPI (blue). GST-3F served as a control. The white scale bar is 10 µm. b) NOCT fractionates with the mitochondria, as tested by western blotting of subcellular fractions (including total, cytosol, and mitochondria) prepared from HEK293T cells that were transfected with NOCT (1-431)-3F. The mitochondrial fractions were divided into three equal aliquots and were either left untreated or were treated with Proteinase K or Proteinase K with Triton X-100. The fractions were then analyzed by SDS PAGE and western blotting with the indicated antibodies. NOCT was detected using guinea pig polyclonal anti-NOCT and goat monoclonal anti-DDDDK (FLAG) antibodies. Fractionation was validated using the following antibodies: anti-uL4m and anti-uS15m for the mitochondrial matrix, anti-Mitofusin 2 and anti-TOM20 for the outer mitochondrial membrane, anti-Histone H3 for the nucleus, and anti-uL1 for the cytoplasm.

    Journal: bioRxiv

    Article Title: Differential processing and localization of human Nocturnin controls metabolism of mRNA and nicotinamide dinucleotide metabolites

    doi: 10.1101/2020.01.12.903534

    Figure Lengend Snippet: NOCT protein is localized to the mitochondria, dependent on its N-terminal mitochondrial targeting sequence. a) Immunofluorescence analysis of intracellular localization of NOCT constructs. NOCT-3F is localized to the mitochondria whereas NOCT Δ( 2 - 15 )-3F and NOCT Δ( 2 - 67 )-3F constructs, which lack the MTS, are predominantly localized to the cytoplasm. The NOCT expression plasmids indicated on the left were transfected into 143B human osteosarcoma cells and visualized using anti-FLAG monoclonal antibody immunofluorescence against the C-terminal 3x-FLAG epitope on each construct with Alexa Fluor Plus 488 secondary antibody (green). Mitochondria are visualized with MitoTracker Red CMXRos (red) and nuclei are visualized with DAPI (blue). GST-3F served as a control. The white scale bar is 10 µm. b) NOCT fractionates with the mitochondria, as tested by western blotting of subcellular fractions (including total, cytosol, and mitochondria) prepared from HEK293T cells that were transfected with NOCT (1-431)-3F. The mitochondrial fractions were divided into three equal aliquots and were either left untreated or were treated with Proteinase K or Proteinase K with Triton X-100. The fractions were then analyzed by SDS PAGE and western blotting with the indicated antibodies. NOCT was detected using guinea pig polyclonal anti-NOCT and goat monoclonal anti-DDDDK (FLAG) antibodies. Fractionation was validated using the following antibodies: anti-uL4m and anti-uS15m for the mitochondrial matrix, anti-Mitofusin 2 and anti-TOM20 for the outer mitochondrial membrane, anti-Histone H3 for the nucleus, and anti-uL1 for the cytoplasm.

    Article Snippet: Mitochondrial fractions were subjected to no treatment, treatment with Proteinase K (20 μg/4 mg mitochondrial protein, Thermo Fisher), or treatment with Proteinase K + 1% Triton X-100 to expose intramitochondrial proteins to protease activity.

    Techniques: Sequencing, Immunofluorescence, Construct, Expressing, Transfection, FLAG-tag, Western Blot, SDS Page, Fractionation

    Layout of the CLASP. Graphic depiction of the CLASP method. (1) Cells of interest are crosslinked with a crosslinker of choice. (2) Small fragments of chromatin are generated by mechanical shearing of the fixed cells. (3) Recombinant dCas9-3×FLAG loaded with the chosen guide RNA is added to the chromatin mixture. (4) Anti-FLAG antibody conjugated to resin is added to RNP/chromatin and washed; enriched chromatin is eluted with 3×FLAG peptide. (5) Through the use of either Proteinase K or nuclease treatment, enriched DNA or protein samples can be isolated for downstream applications.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: dCas9-targeted locus-specific protein isolation method identifies histone gene regulators

    doi: 10.1073/pnas.1718844115

    Figure Lengend Snippet: Layout of the CLASP. Graphic depiction of the CLASP method. (1) Cells of interest are crosslinked with a crosslinker of choice. (2) Small fragments of chromatin are generated by mechanical shearing of the fixed cells. (3) Recombinant dCas9-3×FLAG loaded with the chosen guide RNA is added to the chromatin mixture. (4) Anti-FLAG antibody conjugated to resin is added to RNP/chromatin and washed; enriched chromatin is eluted with 3×FLAG peptide. (5) Through the use of either Proteinase K or nuclease treatment, enriched DNA or protein samples can be isolated for downstream applications.

    Article Snippet: Twenty units of DnaseI (New England Biolabs) were added to the washed resin in 1× NT2 buffer with 150 mM NaCl and incubated at 37 °C for 30 min. SDS was added to mixture to get a 0.1% final concentration, and the mixture was treated with 2.5 uL of Proteinase K (Thermo Fisher) at 56 °C for 1 h. RNA was isolated by using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols. cDNA synthesis was performed with 50 ug of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad) and was diluted 10-fold.

    Techniques: Generated, Recombinant, Isolation

    ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.

    Journal: Prion

    Article Title: Prion Pathogenesis is Independent of Caspase-12

    doi:

    Figure Lengend Snippet: ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.

    Article Snippet: For proteinase K treatment, 10% brain homogenates from terminally ill WT or Caspase-12 KOs were diluted to 1% in lysis buffer and treated with 50 µg/ml proteinase K (Invitrogen) for one hour at 37°C.

    Techniques: Infection, Mouse Assay, Western Blot, Migration

    Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.

    Journal: Prion

    Article Title: Prion Pathogenesis is Independent of Caspase-12

    doi:

    Figure Lengend Snippet: Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.

    Article Snippet: For proteinase K treatment, 10% brain homogenates from terminally ill WT or Caspase-12 KOs were diluted to 1% in lysis buffer and treated with 50 µg/ml proteinase K (Invitrogen) for one hour at 37°C.

    Techniques: Mouse Assay, Staining

    Morphological apoptosis of human neutrophils incubated in vitro with proteinase K- or periodate-treated purified HGE agent. Treatment of purified HGE agent with proteinase K completely eliminated the antiapoptotic effect of the purified HGE agent. However, periodate treatment did not change the antiapoptotic effect of the purified HGE agent. The results were significantly (∗, P

    Journal: Infection and Immunity

    Article Title: Intracellular Infection by the Human Granulocytic Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis

    doi:

    Figure Lengend Snippet: Morphological apoptosis of human neutrophils incubated in vitro with proteinase K- or periodate-treated purified HGE agent. Treatment of purified HGE agent with proteinase K completely eliminated the antiapoptotic effect of the purified HGE agent. However, periodate treatment did not change the antiapoptotic effect of the purified HGE agent. The results were significantly (∗, P

    Article Snippet: For proteinase K treatment, the purified HGE agent was incubated in 1 mg of proteinase K (GIBCO)/ml in distilled water at 60°C for 2 h. After incubation, 1 mM phenylmethylsulfonyl fluoride (Sigma) was added, the mixture was incubated for 10 min at 60°C, and then the ehrlichiae were washed three times in RPMI 1640 medium ( , ).

    Techniques: Incubation, In Vitro, Purification