proteinase k Search Results


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  • 99
    New England Biolabs proteinase k
    Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher proteinase k
    Proteinase K, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore proteinase k
    Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 26156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen proteinase k
    Proteinase K, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 20981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies proteinase k
    Ubiquitin and α-syn aggregates colocalize with neuronal markers. Sections of hippocampus in 12-mo-old Gba1 D409V/D409V brains, either pretreated with <t>proteinase</t> K (PK) ( B ) or not ( A and C ) before immunostaining. Immunostaining for NFH ( A ) exposed
    Proteinase K, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 4790 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore proteinase inhibitor cocktail
    Ubiquitin and α-syn aggregates colocalize with neuronal markers. Sections of hippocampus in 12-mo-old Gba1 D409V/D409V brains, either pretreated with <t>proteinase</t> K (PK) ( B ) or not ( A and C ) before immunostaining. Immunostaining for NFH ( A ) exposed
    Proteinase Inhibitor Cocktail, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3974 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim proteinase k
    Treatment of ScN2a cells with PPI. ScN2a cells were transfected transiently with the pSPOX expression vector encoding either the full-length MHM2 or the MHM2(Δ23–88,Δ141–221) sequence. Three days after transfection cells were incubated with either control medium or medium plus 150 μg of PPI generation 4.0 per ml for 4 h. Cells were then harvested and lysed as described in Materials and Methods. Minus symbols denote undigested control lysate, and plus symbols designate the pellet fraction of lysate subjected to limited proteolysis with 20 μg of <t>proteinase</t> K per ml for 1 h at 37°C, corresponding to a total protein/proteinase K ratio of 25:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons. Lane 1, MHM2 control; lane 2, MHM2 plus PPI; lane 3, PrP61 control; lane 4, PrP61 plus PPI.
    Proteinase K, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 2692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher proteinase k solution
    Treatment of ScN2a cells with PPI. ScN2a cells were transfected transiently with the pSPOX expression vector encoding either the full-length MHM2 or the MHM2(Δ23–88,Δ141–221) sequence. Three days after transfection cells were incubated with either control medium or medium plus 150 μg of PPI generation 4.0 per ml for 4 h. Cells were then harvested and lysed as described in Materials and Methods. Minus symbols denote undigested control lysate, and plus symbols designate the pellet fraction of lysate subjected to limited proteolysis with 20 μg of <t>proteinase</t> K per ml for 1 h at 37°C, corresponding to a total protein/proteinase K ratio of 25:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons. Lane 1, MHM2 control; lane 2, MHM2 plus PPI; lane 3, PrP61 control; lane 4, PrP61 plus PPI.
    Proteinase K Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa proteinase k
    Treatment of ScN2a cells with PPI. ScN2a cells were transfected transiently with the pSPOX expression vector encoding either the full-length MHM2 or the MHM2(Δ23–88,Δ141–221) sequence. Three days after transfection cells were incubated with either control medium or medium plus 150 μg of PPI generation 4.0 per ml for 4 h. Cells were then harvested and lysed as described in Materials and Methods. Minus symbols denote undigested control lysate, and plus symbols designate the pellet fraction of lysate subjected to limited proteolysis with 20 μg of <t>proteinase</t> K per ml for 1 h at 37°C, corresponding to a total protein/proteinase K ratio of 25:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons. Lane 1, MHM2 control; lane 2, MHM2 plus PPI; lane 3, PrP61 control; lane 4, PrP61 plus PPI.
    Proteinase K, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1759 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA proteinase k
    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
    Proteinase K, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 1011 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co proteinase k
    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
    Proteinase K, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM proteinase k
    The effect of <t>Proteinase</t> K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C
    Proteinase K, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 833 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific proteinase k
    Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) <t>Proteinase</t> K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.
    Proteinase K, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 788 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad proteinase k
    Characterization of RiCF. (A)  Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone.  (B)  Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM.  (C)  RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE.  (D)  Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented.  (E)  A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis.  (F)  Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.
    Proteinase K, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco proteinase k
    In vivo cross-linking of telomeres. Mycelium from liquid cultures was treated with DSS or DSG. Total DNA was isolated, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized to labeled plasmid DNA. ( A ) pLUS891L. The physical map of the plasmid DNA is shown above. The sizes of the restriction fragments are indicated (in kb). tsr , thiostrepton resistance gene; ARS, autonomously replicating sequence of pSLA2; filled arrows, SCP1 telomeres; filled circles, Tpc proteins. The DNA linked by the cross-linker is indicated by an asterisk. ‘M’, DNA size markers. The size of the DNA fragments is depicted on the left (in kb). A set of samples was treated with <t>proteinase</t> K (‘+’) before electrophoresis (right panel). ( B ) pLUS892L. The symbols and analyses are as in (A).
    Proteinase K, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 1044 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore proteinase k solution
    In vivo cross-linking of telomeres. Mycelium from liquid cultures was treated with DSS or DSG. Total DNA was isolated, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized to labeled plasmid DNA. ( A ) pLUS891L. The physical map of the plasmid DNA is shown above. The sizes of the restriction fragments are indicated (in kb). tsr , thiostrepton resistance gene; ARS, autonomously replicating sequence of pSLA2; filled arrows, SCP1 telomeres; filled circles, Tpc proteins. The DNA linked by the cross-linker is indicated by an asterisk. ‘M’, DNA size markers. The size of the DNA fragments is depicted on the left (in kb). A set of samples was treated with <t>proteinase</t> K (‘+’) before electrophoresis (right panel). ( B ) pLUS892L. The symbols and analyses are as in (A).
    Proteinase K Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant proteinase k
    (page width). Schematic illustrating the steps in the CHP microplate assay. (A) After incubation in F-CHP, the sample is rinsed, removing unbound F-CHP. (B) The sample is digested with <t>proteinase</t> K to homogenize it. (C) The fluorescence of the digest is quantified on a 96-well microplate reader.
    Proteinase K, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Carl Roth GmbH proteinase k
    Evidence for distinct circRNA-protein complexes in mammalian cells. ( A ) Sedimentation profiles of circRNPs/circRNAs. Cytoplasmic S100 extract and corresponding free RNA from HeLa cells were fractionated by glycerol gradient centrifugation (#1–22 from top to bottom; the last fraction containing the resuspended pellet), followed by RT-PCR analysis of 12 abundant circRNAs across the gradient (ordered from top to bottom according to their sizes; given in nucleotides). The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). ( B ) Quantitation of circRNA distribution across gradient, comparing free RNA prepared from extract (RNA, in blue), cytoplasmic S100 fraction (extract, in red), and proteinase K-treated extract (extract + PK, in dashed lines). Data from panel A (RNA/extract) and from  Supplementary Fig. S1A  (extract + PK) were used for quantitation.
    Proteinase K, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    A serine protease that displays the ability to digest native proteins thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process It retains its activity in
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    N/A
    A serine protease that displays the ability to digest native proteins thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process It retains its activity in
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    N/A
    A serine protease that displays the ability to digest native proteins thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process It retains its activity in
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    N/A
    A serine protease that displays the ability to digest native proteins thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process It retains its activity in
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    Image Search Results


    Ubiquitin and α-syn aggregates colocalize with neuronal markers. Sections of hippocampus in 12-mo-old Gba1 D409V/D409V brains, either pretreated with proteinase K (PK) ( B ) or not ( A and C ) before immunostaining. Immunostaining for NFH ( A ) exposed

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

    Article Title: CNS expression of glucocerebrosidase corrects ?-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy

    doi: 10.1073/pnas.1108197108

    Figure Lengend Snippet: Ubiquitin and α-syn aggregates colocalize with neuronal markers. Sections of hippocampus in 12-mo-old Gba1 D409V/D409V brains, either pretreated with proteinase K (PK) ( B ) or not ( A and C ) before immunostaining. Immunostaining for NFH ( A ) exposed

    Article Snippet: Some tissue sections were pretreated with proteinase K (1:4 dilution; DAKO) for 7 min at room temperature to expose α-syn and other aggregated proteins ( ).

    Techniques: Immunostaining

    Progressive accumulation of α-syn/ubiquitin aggregates in Gba1 D409V/D409V Gaucher mouse brains. ( A ) Frozen sections were pretreated without ( Upper ) or with ( Lower ) proteinase K (PK) to uncover endogenous α-syn aggregates (arrowheads).

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

    Article Title: CNS expression of glucocerebrosidase corrects ?-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy

    doi: 10.1073/pnas.1108197108

    Figure Lengend Snippet: Progressive accumulation of α-syn/ubiquitin aggregates in Gba1 D409V/D409V Gaucher mouse brains. ( A ) Frozen sections were pretreated without ( Upper ) or with ( Lower ) proteinase K (PK) to uncover endogenous α-syn aggregates (arrowheads).

    Article Snippet: Some tissue sections were pretreated with proteinase K (1:4 dilution; DAKO) for 7 min at room temperature to expose α-syn and other aggregated proteins ( ).

    Techniques:

    Treatment of ScN2a cells with PPI. ScN2a cells were transfected transiently with the pSPOX expression vector encoding either the full-length MHM2 or the MHM2(Δ23–88,Δ141–221) sequence. Three days after transfection cells were incubated with either control medium or medium plus 150 μg of PPI generation 4.0 per ml for 4 h. Cells were then harvested and lysed as described in Materials and Methods. Minus symbols denote undigested control lysate, and plus symbols designate the pellet fraction of lysate subjected to limited proteolysis with 20 μg of proteinase K per ml for 1 h at 37°C, corresponding to a total protein/proteinase K ratio of 25:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons. Lane 1, MHM2 control; lane 2, MHM2 plus PPI; lane 3, PrP61 control; lane 4, PrP61 plus PPI.

    Journal: Molecular and Cellular Biology

    Article Title: A Protease-Resistant 61-Residue Prion Peptide Causes Neurodegeneration in Transgenic Mice

    doi: 10.1128/MCB.21.7.2608-2616.2001

    Figure Lengend Snippet: Treatment of ScN2a cells with PPI. ScN2a cells were transfected transiently with the pSPOX expression vector encoding either the full-length MHM2 or the MHM2(Δ23–88,Δ141–221) sequence. Three days after transfection cells were incubated with either control medium or medium plus 150 μg of PPI generation 4.0 per ml for 4 h. Cells were then harvested and lysed as described in Materials and Methods. Minus symbols denote undigested control lysate, and plus symbols designate the pellet fraction of lysate subjected to limited proteolysis with 20 μg of proteinase K per ml for 1 h at 37°C, corresponding to a total protein/proteinase K ratio of 25:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons. Lane 1, MHM2 control; lane 2, MHM2 plus PPI; lane 3, PrP61 control; lane 4, PrP61 plus PPI.

    Article Snippet: Proteinase K (Boehringer Mannheim) was added to 0.5 ml of adjusted homogenate to achieve a ratio of total protein to enzyme of 50:1.

    Techniques: Transfection, Expressing, Plasmid Preparation, Sequencing, Incubation, Migration

    Expression of PrP deletion constructs in scrapie-infected neuroblastoma cells. Scrapie-infected (ScN2a) cells were transfected transiently with the pSPOX expression vector carrying modified PrP genes as noted below. Lane 1, MHM2; lane 2, MHM2(Δ23–88); lane 3, MHM2(Δ23–88,Δ141–176); lane 4, MHM2(Δ23–88,Δ127–176); lane 5, MHM2(Δ23–88,Δ127–164); lane 6, mock transfection. Minus symbols denote undigested control sample, and plus symbols designate the pellet fraction of sample subjected to limited proteolysis with 7 μg of proteinase K per ml for 30 min at 37°C, corresponding to a total protein/proteinase K ratio of 71:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: A Protease-Resistant 61-Residue Prion Peptide Causes Neurodegeneration in Transgenic Mice

    doi: 10.1128/MCB.21.7.2608-2616.2001

    Figure Lengend Snippet: Expression of PrP deletion constructs in scrapie-infected neuroblastoma cells. Scrapie-infected (ScN2a) cells were transfected transiently with the pSPOX expression vector carrying modified PrP genes as noted below. Lane 1, MHM2; lane 2, MHM2(Δ23–88); lane 3, MHM2(Δ23–88,Δ141–176); lane 4, MHM2(Δ23–88,Δ127–176); lane 5, MHM2(Δ23–88,Δ127–164); lane 6, mock transfection. Minus symbols denote undigested control sample, and plus symbols designate the pellet fraction of sample subjected to limited proteolysis with 7 μg of proteinase K per ml for 30 min at 37°C, corresponding to a total protein/proteinase K ratio of 71:1. Units are apparent molecular sizes based on migration of protein standards in kilodaltons.

    Article Snippet: Proteinase K (Boehringer Mannheim) was added to 0.5 ml of adjusted homogenate to achieve a ratio of total protein to enzyme of 50:1.

    Techniques: Expressing, Construct, Infection, Transfection, Plasmid Preparation, Modification, Migration

    ). None, no fragments detected with MAb 3F4 after digestion with 7 μg of proteinase K per ml for 30 min at 37°C; ++, fragments detected after digestion with 7 μg of proteinase K per ml for 30 min at 37°C but not after digestion with 20 μg of proteinase K per ml for 1 h at 37°C; ++++, intense 3F4 immunoreactive fragments seen even after digestion with 20 μg of proteinase K per ml for 1 h at 37°C. HA, α-helix A; HB, α-helix B; HC, α-helix C; CHO, carbohydrate; S-S, disulfide bond.

    Journal: Molecular and Cellular Biology

    Article Title: A Protease-Resistant 61-Residue Prion Peptide Causes Neurodegeneration in Transgenic Mice

    doi: 10.1128/MCB.21.7.2608-2616.2001

    Figure Lengend Snippet: ). None, no fragments detected with MAb 3F4 after digestion with 7 μg of proteinase K per ml for 30 min at 37°C; ++, fragments detected after digestion with 7 μg of proteinase K per ml for 30 min at 37°C but not after digestion with 20 μg of proteinase K per ml for 1 h at 37°C; ++++, intense 3F4 immunoreactive fragments seen even after digestion with 20 μg of proteinase K per ml for 1 h at 37°C. HA, α-helix A; HB, α-helix B; HC, α-helix C; CHO, carbohydrate; S-S, disulfide bond.

    Article Snippet: Proteinase K (Boehringer Mannheim) was added to 0.5 ml of adjusted homogenate to achieve a ratio of total protein to enzyme of 50:1.

    Techniques:

    Protease digestion of MHM2(Δ23–88,Δ141–221). (A) Lanes A1 through A3, N2a cells transfected with pSPOX[MHM2(Δ23–88,Δ141–221)]; lanes B1 through B3, mock-transfected N2a cells; lanes C1 through C3, 0.4 μg of myristylated, synthetic MHM2(Δ23–88,Δ141–221) peptide per ml mixed with untransfected N2a cell lysate (0.5 mg of total protein per ml). Lanes A1, B1, and C1, untreated whole-cell lysates; lanes A2, B2, and C2, pellet fractions of lysates digested with 7 μg of proteinase K per ml for 30 min at 37°C; lanes A3, B3, and C3, pellet fractions of lysates digested with 20 μg of proteinase K per ml for 1 h at 37°C. SDS-PAGE was performed on 16% Tricine gels (Novex). Western blotting was performed with 3F4 MAb at 1:5,000 dilution. After processing, lanes 1 and 2 were exposed for 1 min and lane 3 was exposed for 15 min to Hypermax film (Amersham Life Sciences). Units are apparent molecular sizes based on migration of protein standards in kilodaltons. (B) Proteinase K digestion of brain homogenates from Tg mice. Lane 1, 60-day-old, uninoculated Tg(PrP106)Prnp 0/0 mouse; lane 2, 65-day-old, scrapie-infected Tg(PrP106)Prnp 0/0 mouse; lane 3, 48-day-old, spontaneously ataxic Tg(PrP61)Prnp 0/0 mouse. Minus symbols denote undigested control sample, and plus symbols designate the pellet fraction of sample subjected to digestion with 20 μg of proteinase K per ml for 1 h at 37°C. Units are apparent molecular sizes based on migration of protein standards in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: A Protease-Resistant 61-Residue Prion Peptide Causes Neurodegeneration in Transgenic Mice

    doi: 10.1128/MCB.21.7.2608-2616.2001

    Figure Lengend Snippet: Protease digestion of MHM2(Δ23–88,Δ141–221). (A) Lanes A1 through A3, N2a cells transfected with pSPOX[MHM2(Δ23–88,Δ141–221)]; lanes B1 through B3, mock-transfected N2a cells; lanes C1 through C3, 0.4 μg of myristylated, synthetic MHM2(Δ23–88,Δ141–221) peptide per ml mixed with untransfected N2a cell lysate (0.5 mg of total protein per ml). Lanes A1, B1, and C1, untreated whole-cell lysates; lanes A2, B2, and C2, pellet fractions of lysates digested with 7 μg of proteinase K per ml for 30 min at 37°C; lanes A3, B3, and C3, pellet fractions of lysates digested with 20 μg of proteinase K per ml for 1 h at 37°C. SDS-PAGE was performed on 16% Tricine gels (Novex). Western blotting was performed with 3F4 MAb at 1:5,000 dilution. After processing, lanes 1 and 2 were exposed for 1 min and lane 3 was exposed for 15 min to Hypermax film (Amersham Life Sciences). Units are apparent molecular sizes based on migration of protein standards in kilodaltons. (B) Proteinase K digestion of brain homogenates from Tg mice. Lane 1, 60-day-old, uninoculated Tg(PrP106)Prnp 0/0 mouse; lane 2, 65-day-old, scrapie-infected Tg(PrP106)Prnp 0/0 mouse; lane 3, 48-day-old, spontaneously ataxic Tg(PrP61)Prnp 0/0 mouse. Minus symbols denote undigested control sample, and plus symbols designate the pellet fraction of sample subjected to digestion with 20 μg of proteinase K per ml for 1 h at 37°C. Units are apparent molecular sizes based on migration of protein standards in kilodaltons.

    Article Snippet: Proteinase K (Boehringer Mannheim) was added to 0.5 ml of adjusted homogenate to achieve a ratio of total protein to enzyme of 50:1.

    Techniques: Transfection, SDS Page, Western Blot, Migration, Mouse Assay, Infection

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2019.02568

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

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2019.02568

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

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2019.02568

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

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2019.02568

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

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

    The effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

    Journal: BMC Research Notes

    Article Title: Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution

    doi: 10.1186/s13104-018-3914-4

    Figure Lengend Snippet: The effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed K1 cells. RNA was extracted from filter-trapped K1 cells after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

    Article Snippet: The filters with the trapped cells were washed with PBS and incubated in 500 μl lysis buffer [10 mM Tris–HCl (pH 7.8), 5 mM EDTA and 0.5% SDS] with or without 400 mg/ml Proteinase K (Wako) at 55 °C.

    Techniques: Incubation

    Effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed thyroid FNAB specimens. The thyroid FNAB specimens processed for LBC using CytoRich-Red solution were trapped to filters in the same way as described above. RNA was extracted from the specimens after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

    Journal: BMC Research Notes

    Article Title: Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution

    doi: 10.1186/s13104-018-3914-4

    Figure Lengend Snippet: Effect of Proteinase K treatment on RNA recovery from CytoRichRed-fixed thyroid FNAB specimens. The thyroid FNAB specimens processed for LBC using CytoRich-Red solution were trapped to filters in the same way as described above. RNA was extracted from the specimens after incubation with Proteinase K-containing and Proteinase K-free buffers for 3 h at 55 °C

    Article Snippet: The filters with the trapped cells were washed with PBS and incubated in 500 μl lysis buffer [10 mM Tris–HCl (pH 7.8), 5 mM EDTA and 0.5% SDS] with or without 400 mg/ml Proteinase K (Wako) at 55 °C.

    Techniques: Incubation

    RNA recovery from filter-trapped K1 cells fixed for liquid-based cytology. RNA was extracted from the fixed K1 cells after incubation with Proteinase K-containing for the indicated periods at 55 °C. RNA recovery from K1 cells fixed with CytoRich-Red ( a ) and CytoRich-Blue ( b ). Black bars and gray bars represent the RNA yield from 20,000 to 100,000 cells, respectively. Quality of RNAs extracted from K1 cells fixed with CytoRich-Red ( c ) and CytoRich-Blue ( d ). The analysis using Agilent 2100 bioanalyzer revealed that 28S and 18S ribosomal RNAs (correspond to the bands at approximately 1900 nt and 3900 nt on the charts, respectively) remained nearly intact, leading to high RINs.  N/A  not assessed

    Journal: BMC Research Notes

    Article Title: Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution

    doi: 10.1186/s13104-018-3914-4

    Figure Lengend Snippet: RNA recovery from filter-trapped K1 cells fixed for liquid-based cytology. RNA was extracted from the fixed K1 cells after incubation with Proteinase K-containing for the indicated periods at 55 °C. RNA recovery from K1 cells fixed with CytoRich-Red ( a ) and CytoRich-Blue ( b ). Black bars and gray bars represent the RNA yield from 20,000 to 100,000 cells, respectively. Quality of RNAs extracted from K1 cells fixed with CytoRich-Red ( c ) and CytoRich-Blue ( d ). The analysis using Agilent 2100 bioanalyzer revealed that 28S and 18S ribosomal RNAs (correspond to the bands at approximately 1900 nt and 3900 nt on the charts, respectively) remained nearly intact, leading to high RINs. N/A not assessed

    Article Snippet: The filters with the trapped cells were washed with PBS and incubated in 500 μl lysis buffer [10 mM Tris–HCl (pH 7.8), 5 mM EDTA and 0.5% SDS] with or without 400 mg/ml Proteinase K (Wako) at 55 °C.

    Techniques: Incubation

    Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) Proteinase K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.

    Journal: Veterinary Sciences

    Article Title: Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

    doi: 10.3390/vetsci6040088

    Figure Lengend Snippet: Preparation of EtOH extract for mass spectrometry analysis. ( a ) Map bacilli were washed 6 times prior to EtOH extraction (labeled × 6) to avoid bovine serum albumin (BSA) contamination. A second extract was prepared after washing Map once (label × 1). Lipids and carbohydrate were removed from the EtOH extract through a second chloroform:methanol:water extraction. This second extraction was run on 12% SDS-PAGE denaturing gels and exposed to Coomassie stain (CBB) and silver stain. The location of BSA migration is identified for both gel images. ( b ) Proteinase K treatment of Map EtOH extract. Inclusion of proteinase K or the EtOH extract along with staining method is indicated beneath the gel. Kilodalton size markers are shown in the left margins of all gels.

    Article Snippet: In the ELISA experiment, to measure antibody binding, proteinase K (200 µg/mL; ACROS Organics-Thermo Fisher Scientific, Pittsburgh, PA, USA) was used.

    Techniques: Mass Spectrometry, Labeling, SDS Page, Staining, Silver Staining, Migration

    Bovine antibody binds to a carbohydrate component of the Map EtOH extract, not to protein. ( a ) Surface antigens of Map K-10 were extracted with 80% ethanol (EtOH Whole) and fractionated by Folch extraction method into organic (chloroform), interface and aqueous fractions. After evaporating methanol for immobilization of the lipids (and other molecules) onto the wells of a microtitre plate, they were incubated with serum samples (1:100 dilution) collected from JD-positive (solid bar) and negative (open bar) cattle. Histogram bars represent mean antibody binding ± standard deviation of quadruplicate determinations. Most of the antigenicity is in the organic fraction. ( b ) EtOH extract treated with proteases shows no negative effects on bovine antibody binding. The extract was treated with either 10 µg/mL trypsin or 10 µg/mL proteinase K with each treatment actually enhancing antibody binding. This enhancement was not statistically significant. However, EtOH extract antigens are efficiently removed by ConA-agarose as shown by lack of antibody binding ( c ). Absorption with agarose only does not affect antibody binding to the EtOH extract. This experiment was repeated in triplicate with qraduplicate measurements for each. p values less than 0.01 are denoted by an asterisk.

    Journal: Veterinary Sciences

    Article Title: Characterization of Ethanol Extracted Cell Wall Components of Mycobacterium avium Subsp. paratuberculosis

    doi: 10.3390/vetsci6040088

    Figure Lengend Snippet: Bovine antibody binds to a carbohydrate component of the Map EtOH extract, not to protein. ( a ) Surface antigens of Map K-10 were extracted with 80% ethanol (EtOH Whole) and fractionated by Folch extraction method into organic (chloroform), interface and aqueous fractions. After evaporating methanol for immobilization of the lipids (and other molecules) onto the wells of a microtitre plate, they were incubated with serum samples (1:100 dilution) collected from JD-positive (solid bar) and negative (open bar) cattle. Histogram bars represent mean antibody binding ± standard deviation of quadruplicate determinations. Most of the antigenicity is in the organic fraction. ( b ) EtOH extract treated with proteases shows no negative effects on bovine antibody binding. The extract was treated with either 10 µg/mL trypsin or 10 µg/mL proteinase K with each treatment actually enhancing antibody binding. This enhancement was not statistically significant. However, EtOH extract antigens are efficiently removed by ConA-agarose as shown by lack of antibody binding ( c ). Absorption with agarose only does not affect antibody binding to the EtOH extract. This experiment was repeated in triplicate with qraduplicate measurements for each. p values less than 0.01 are denoted by an asterisk.

    Article Snippet: In the ELISA experiment, to measure antibody binding, proteinase K (200 µg/mL; ACROS Organics-Thermo Fisher Scientific, Pittsburgh, PA, USA) was used.

    Techniques: Incubation, Binding Assay, Standard Deviation

    Characterization of RiCF. (A)  Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone.  (B)  Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM.  (C)  RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE.  (D)  Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented.  (E)  A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis.  (F)  Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Characterization of RiCF. (A) Representative radiogram showing RNase dose dependent chromosomal fragmentation in AB1157. Plugs were made with 0, 2, 10, 25, 50 or 100 μg RNase and lysed and electrophoresed under standard conditions. CZ, compression zone. (B) Quantification showing increase in chromosomal fragmentation in RNase dose-dependent manner. Data points are means of six independent assays ± SEM. (C) RNase-effect is not seen in the pre-lyzed cells. Plugs from AB1157 culture were made in the presence of proteinase K, but without any RNase. After overnight lysis and extensive washing, the plugs were incubated with 0, 2, 20 and 100 μg RNase or 100 U of EcoRI for 15 H at 37°C before PFGE. (D) Quantification of chromosomal fragmentation showing extreme sensitivity of chromosomes to EcoRI, but not RNase, when plugs were treated with the enzymes after lysis of cells. The experiment is done twice and a representative result is presented. (E) A representative radiogram showing kinetics of RiCF. Multiple plugs were made in the presence of RNase (50 μg/plug) and incubated at 62°C for 10, 30, 60, 180 or 900 minutes with lysis buffer in individual tubes. At the indicated times, one tube was removed, lysis buffer was replaced with ice-cold TE, and plugs were stored at 4°C until all plugs were ready for electrophoresis. (F) Quantification of kinetics of chromosomal fragmentation when plugs were made in the presence of RNase and lysed for 1, 5, 10, 30, 60, 180 or 900 minutes. Data points are means of three independent assays ± SEM. Arrow shows the value of fragmentation after 10 min lysis.

    Article Snippet: To make agarose plugs, 5 μl of proteinase K (from 5 mg/ml stock) and 65 μl of molten and cooled to 75°C agarose (either plain or lysis-plug agarose) was added to the cell suspension, and the mixture was quickly transferred to plug molds (Bio-Rad).

    Techniques: Lysis, Incubation, Electrophoresis

    Genetics of RiCF. (A)  Quantitative determination of RiCF in Δ hns  mutant. AB1157 and SRK254 were grown at 37°C to the same final OD, and plugs were made in the absence of proteinase K, but with or without RNase (50 μg/plug). After overnight incubation in the lysis buffer at 62°C, the plugs were electrophoresed under standard conditions. Data points are means of 6–10 independent assays± SEM.  (B)  Radiogram of a representative pulsed field gel from which data in (A) are calculated.  (C)  Comparison of RiCF in Δ hupA  Δ hupB  and Δ ihfA  Δ ihfB  double mutants. Experiment was done as described in (A), and values presented are means of 6–13 independent assays ± SEM.  (D)  Radiogram of a representative pulsed field gel from which data in (C) are calculated.  (E)  Effect of  hns  deletion on RiCF of Δ ihfA  Δ ihfB  mutant. Values presented are means of 7 independent assays ± SEM.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Genetics of RiCF. (A) Quantitative determination of RiCF in Δ hns mutant. AB1157 and SRK254 were grown at 37°C to the same final OD, and plugs were made in the absence of proteinase K, but with or without RNase (50 μg/plug). After overnight incubation in the lysis buffer at 62°C, the plugs were electrophoresed under standard conditions. Data points are means of 6–10 independent assays± SEM. (B) Radiogram of a representative pulsed field gel from which data in (A) are calculated. (C) Comparison of RiCF in Δ hupA Δ hupB and Δ ihfA Δ ihfB double mutants. Experiment was done as described in (A), and values presented are means of 6–13 independent assays ± SEM. (D) Radiogram of a representative pulsed field gel from which data in (C) are calculated. (E) Effect of hns deletion on RiCF of Δ ihfA Δ ihfB mutant. Values presented are means of 7 independent assays ± SEM.

    Article Snippet: To make agarose plugs, 5 μl of proteinase K (from 5 mg/ml stock) and 65 μl of molten and cooled to 75°C agarose (either plain or lysis-plug agarose) was added to the cell suspension, and the mixture was quickly transferred to plug molds (Bio-Rad).

    Techniques: Mutagenesis, Incubation, Lysis, Pulsed-Field Gel

    Non-coding RNA and HU stabilize nucleoids. (A)  Comparison of spontaneous and RNase-induced fragmentation in Δ nc1  Δ nc5  Δ ihfA  Δ ihfB , Δ nc1  Δ nc5  Δ hupA  Δ hupB  and Δ nc1  Δ nc5  Δ hns  mutants. AB1157, SRK254-12, SRK254-15 and SRK254-18 were grown at 37°C, and plugs were made in absence of proteinase K, both with or without RNase (50 μg/plug), and lysed under standard conditions. Data points are means of at least three independent assays± SEM.  (B)  Radiogram of a representative gel from which data in (A) are generated.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Non-coding RNA and HU stabilize nucleoids. (A) Comparison of spontaneous and RNase-induced fragmentation in Δ nc1 Δ nc5 Δ ihfA Δ ihfB , Δ nc1 Δ nc5 Δ hupA Δ hupB and Δ nc1 Δ nc5 Δ hns mutants. AB1157, SRK254-12, SRK254-15 and SRK254-18 were grown at 37°C, and plugs were made in absence of proteinase K, both with or without RNase (50 μg/plug), and lysed under standard conditions. Data points are means of at least three independent assays± SEM. (B) Radiogram of a representative gel from which data in (A) are generated.

    Article Snippet: To make agarose plugs, 5 μl of proteinase K (from 5 mg/ml stock) and 65 μl of molten and cooled to 75°C agarose (either plain or lysis-plug agarose) was added to the cell suspension, and the mixture was quickly transferred to plug molds (Bio-Rad).

    Techniques: Generated

    Endonuclease-I is critical for RiCF but not for spontaneous fragmentation. (A)  Comparison of spontaneous fragmentation and RiCF in AB1157, AB1157 Δ nc1  Δ nc5  Δ hupA  Δ hupB  and AB1157 Δ nc1  Δ nc5  Δ hupA  Δ hupB  Δ endA  mutants. All strains were grown at 37°C to the final OD of 0.6, and plugs were made in the absence of proteinase K, both with or without RNase (50 μg/plug). Data points are means of at least three independent assays ± SEM.  (B)  Radiogram of a representative gel from which data in (A) are generated.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Endonuclease-I is critical for RiCF but not for spontaneous fragmentation. (A) Comparison of spontaneous fragmentation and RiCF in AB1157, AB1157 Δ nc1 Δ nc5 Δ hupA Δ hupB and AB1157 Δ nc1 Δ nc5 Δ hupA Δ hupB Δ endA mutants. All strains were grown at 37°C to the final OD of 0.6, and plugs were made in the absence of proteinase K, both with or without RNase (50 μg/plug). Data points are means of at least three independent assays ± SEM. (B) Radiogram of a representative gel from which data in (A) are generated.

    Article Snippet: To make agarose plugs, 5 μl of proteinase K (from 5 mg/ml stock) and 65 μl of molten and cooled to 75°C agarose (either plain or lysis-plug agarose) was added to the cell suspension, and the mixture was quickly transferred to plug molds (Bio-Rad).

    Techniques: Generated

    RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Article Snippet: To make agarose plugs, 5 μl of proteinase K (from 5 mg/ml stock) and 65 μl of molten and cooled to 75°C agarose (either plain or lysis-plug agarose) was added to the cell suspension, and the mixture was quickly transferred to plug molds (Bio-Rad).

    Techniques: Pulsed-Field Gel, Lysis

    In vivo cross-linking of telomeres. Mycelium from liquid cultures was treated with DSS or DSG. Total DNA was isolated, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized to labeled plasmid DNA. ( A ) pLUS891L. The physical map of the plasmid DNA is shown above. The sizes of the restriction fragments are indicated (in kb). tsr , thiostrepton resistance gene; ARS, autonomously replicating sequence of pSLA2; filled arrows, SCP1 telomeres; filled circles, Tpc proteins. The DNA linked by the cross-linker is indicated by an asterisk. ‘M’, DNA size markers. The size of the DNA fragments is depicted on the left (in kb). A set of samples was treated with proteinase K (‘+’) before electrophoresis (right panel). ( B ) pLUS892L. The symbols and analyses are as in (A).

    Journal: Nucleic Acids Research

    Article Title: Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

    doi: 10.1093/nar/gkq1204

    Figure Lengend Snippet: In vivo cross-linking of telomeres. Mycelium from liquid cultures was treated with DSS or DSG. Total DNA was isolated, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized to labeled plasmid DNA. ( A ) pLUS891L. The physical map of the plasmid DNA is shown above. The sizes of the restriction fragments are indicated (in kb). tsr , thiostrepton resistance gene; ARS, autonomously replicating sequence of pSLA2; filled arrows, SCP1 telomeres; filled circles, Tpc proteins. The DNA linked by the cross-linker is indicated by an asterisk. ‘M’, DNA size markers. The size of the DNA fragments is depicted on the left (in kb). A set of samples was treated with proteinase K (‘+’) before electrophoresis (right panel). ( B ) pLUS892L. The symbols and analyses are as in (A).

    Article Snippet: To remove TP, proteinase K (AMRESCO) was added to the loading buffer containing the DNA sample to a final concentration of 20 U/ml before electrophoresis.

    Techniques: In Vivo, Isolation, Nucleic Acid Electrophoresis, Labeling, Plasmid Preparation, Sequencing, Electrophoresis

    In vitro cross-linking of telomeres. ( A ) Without GnHCl treatment. Total DNA of S. lividans 3200/pLUS891L was treated with DSS or DSG, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized with pLUS891L DNA probe. ( B ) With GnHCl treatment. Genomic DNA of S. lividans 3200/pLUS892L was isolated by GnHCl–CsCl gradient centrifugation and treated with DSS or DSG. The treated DNA was digested with BclI or MluI, fractionated by gel electrophoresis and hybridized to the SCP1 terminal DNA probe. The physical map of pLUS892L is shown above with the BclI and MluI (Ml) sites and fragments sizes indicated. In the control experiment (right panel), the samples were treated with proteinase K before electrophoresis. The cross-linked DNA is indicated by an asterisk.

    Journal: Nucleic Acids Research

    Article Title: Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

    doi: 10.1093/nar/gkq1204

    Figure Lengend Snippet: In vitro cross-linking of telomeres. ( A ) Without GnHCl treatment. Total DNA of S. lividans 3200/pLUS891L was treated with DSS or DSG, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized with pLUS891L DNA probe. ( B ) With GnHCl treatment. Genomic DNA of S. lividans 3200/pLUS892L was isolated by GnHCl–CsCl gradient centrifugation and treated with DSS or DSG. The treated DNA was digested with BclI or MluI, fractionated by gel electrophoresis and hybridized to the SCP1 terminal DNA probe. The physical map of pLUS892L is shown above with the BclI and MluI (Ml) sites and fragments sizes indicated. In the control experiment (right panel), the samples were treated with proteinase K before electrophoresis. The cross-linked DNA is indicated by an asterisk.

    Article Snippet: To remove TP, proteinase K (AMRESCO) was added to the loading buffer containing the DNA sample to a final concentration of 20 U/ml before electrophoresis.

    Techniques: In Vitro, Nucleic Acid Electrophoresis, Isolation, Gradient Centrifugation, Electrophoresis

    Superhelical structures formed by linear plasmid DNA. ( A ) S. lividans 3200/pLUS892L mycelium was osmotically lysed. The cell lysate was treated with E. coli Topoisomerase I (‘Topo I’), fractionated by gel electrophoresis and hybridized to the pLUS892L DNA probe. In the control experiments, samples were treated with proteinase K (‘PK’) before electrophoresis, or the topoisomerase treatment was omitted, or both. The sizes of the (proteinase K-treated) linear pLUS892L DNA and the marker DNAs (‘M’) are indicated (in kb). ( B ) AFM examination of isolated DSS-cross-linked linear plasmid DNA (pLUS891L) without proteinase K-treatment (‘−PK’) shows supercoiled DNA structures that are held together by telomere–telomere interactions (left and center images). The center image shows a zoomed-in surface plot of the coiled DNA molecule boxed in the left image. Proteolytic digestion of the DSS-cross-linked DNA by proteinase K (‘+PK’) transformed it into relaxed, linear structures (right). The scale bar is 1 µm.

    Journal: Nucleic Acids Research

    Article Title: Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

    doi: 10.1093/nar/gkq1204

    Figure Lengend Snippet: Superhelical structures formed by linear plasmid DNA. ( A ) S. lividans 3200/pLUS892L mycelium was osmotically lysed. The cell lysate was treated with E. coli Topoisomerase I (‘Topo I’), fractionated by gel electrophoresis and hybridized to the pLUS892L DNA probe. In the control experiments, samples were treated with proteinase K (‘PK’) before electrophoresis, or the topoisomerase treatment was omitted, or both. The sizes of the (proteinase K-treated) linear pLUS892L DNA and the marker DNAs (‘M’) are indicated (in kb). ( B ) AFM examination of isolated DSS-cross-linked linear plasmid DNA (pLUS891L) without proteinase K-treatment (‘−PK’) shows supercoiled DNA structures that are held together by telomere–telomere interactions (left and center images). The center image shows a zoomed-in surface plot of the coiled DNA molecule boxed in the left image. Proteolytic digestion of the DSS-cross-linked DNA by proteinase K (‘+PK’) transformed it into relaxed, linear structures (right). The scale bar is 1 µm.

    Article Snippet: To remove TP, proteinase K (AMRESCO) was added to the loading buffer containing the DNA sample to a final concentration of 20 U/ml before electrophoresis.

    Techniques: Plasmid Preparation, Nucleic Acid Electrophoresis, Electrophoresis, Marker, Isolation, Transformation Assay

    (page width). Schematic illustrating the steps in the CHP microplate assay. (A) After incubation in F-CHP, the sample is rinsed, removing unbound F-CHP. (B) The sample is digested with proteinase K to homogenize it. (C) The fluorescence of the digest is quantified on a 96-well microplate reader.

    Journal: bioRxiv

    Article Title: Microplate Assay for Denatured Collagen using Collagen Hybridizing Peptides

    doi: 10.1101/443242

    Figure Lengend Snippet: (page width). Schematic illustrating the steps in the CHP microplate assay. (A) After incubation in F-CHP, the sample is rinsed, removing unbound F-CHP. (B) The sample is digested with proteinase K to homogenize it. (C) The fluorescence of the digest is quantified on a 96-well microplate reader.

    Article Snippet: CHP Microplate AssayTo enable the detection of F-CHP fluorescence using a microplate reader, samples were homogenized in 500 μL of a 1 mg/mL proteinase K (Cat No. 193981, MP Biomedicals, LLC, Santa Ana, CA) solution in deionized water for three hours at 60°C.

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Fluorescence

    Evidence for distinct circRNA-protein complexes in mammalian cells. ( A ) Sedimentation profiles of circRNPs/circRNAs. Cytoplasmic S100 extract and corresponding free RNA from HeLa cells were fractionated by glycerol gradient centrifugation (#1–22 from top to bottom; the last fraction containing the resuspended pellet), followed by RT-PCR analysis of 12 abundant circRNAs across the gradient (ordered from top to bottom according to their sizes; given in nucleotides). The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). ( B ) Quantitation of circRNA distribution across gradient, comparing free RNA prepared from extract (RNA, in blue), cytoplasmic S100 fraction (extract, in red), and proteinase K-treated extract (extract + PK, in dashed lines). Data from panel A (RNA/extract) and from  Supplementary Fig. S1A  (extract + PK) were used for quantitation.

    Journal: Scientific Reports

    Article Title: CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    doi: 10.1038/srep31313

    Figure Lengend Snippet: Evidence for distinct circRNA-protein complexes in mammalian cells. ( A ) Sedimentation profiles of circRNPs/circRNAs. Cytoplasmic S100 extract and corresponding free RNA from HeLa cells were fractionated by glycerol gradient centrifugation (#1–22 from top to bottom; the last fraction containing the resuspended pellet), followed by RT-PCR analysis of 12 abundant circRNAs across the gradient (ordered from top to bottom according to their sizes; given in nucleotides). The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). ( B ) Quantitation of circRNA distribution across gradient, comparing free RNA prepared from extract (RNA, in blue), cytoplasmic S100 fraction (extract, in red), and proteinase K-treated extract (extract + PK, in dashed lines). Data from panel A (RNA/extract) and from Supplementary Fig. S1A (extract + PK) were used for quantitation.

    Article Snippet: After a 20 min incubation at room temperature, the samples were washed three times with 1 ml of washing buffer (10 mM Tris-HCl pH 7.5, 100/300/600 mM KCl, 2.5 mM MgCl2 , 0.1% Triton X-100), treated with proteinase K (Roth), phenol/chloroform extracted, and ethanol precipitated.

    Techniques: Sedimentation, Gradient Centrifugation, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

    IMP3 protein specifically and stably bound to circRNA family: validation and characterization. ( A ) Validation of IMP3 bound to specific circRNAs. Lysates of HepG2, PANC1, and PATU cells were subjected to immunoprecipitation with anti-IMP3 antibodies (IP), or as mock control, with anti-FLAG antibodies (m). Co-precipitated RNA was purified and assayed by RT-PCR for FTL mRNA (positive control), CAMSAP1 (negative control circRNA), and for the putative IMP3-associated circRNAs CDYL, NFATC3, and ANKRD17. For each of the circRNAs, the linear isoform of the respective gene was tested in addition (circ/lin; 90% of the mock- and IMP3-immunoprecipitates were used in RT-PCR). In addition, 5% of the input material was assayed (I). M, markers (in bp). ( B ) Quantitative immunoprecipitation analysis of IMP3-circRNA association in three cell lines (HepG2, PANC1, and PATU). For the same set of IMP3 circRNA targets and controls as shown in panel A, the immunoprecipitation efficiences were determined by RT-qPCR assays (% of input; statistical deviations based on biological duplicates). ( C ) Sedimentation profiles of IMP3-containing circRNPs. Cytoplasmic S100 extract from HeLa cells (extract), corresponding free RNA (RNA), and proteinase K-treated extract (extract + PK) were fractionated by glycerol gradient centrifugation (#1–22; the last fraction contains the resuspended pellet), followed by RT-PCR analysis of two IMP3-containing circRNAs (NFATC3 and ANKRD17). The relatively low recovery of circRNAs NFATC3 (1298 nt) and especially ANKRD17 (1832 nt), the largest circRNAs analyzed in S100 extract, may be caused by the higher tendency of such large circRNPs to aggregate and form precipitates, which were lost in the pellet fraction. The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). For comparison, the distribution of total IMP3 protein across the gradient was visualized by Western blotting in extract and, as a control, in proteinase K-treated extract. ( D ) IMP3 immunoprecipitation efficiencies of gradient-purified NFATC3 and ANKRD17 circRNPs. HeLa cytoplasmic S100 extract was gradient-fractionated, and NFATC3 and ANKRD17 circRNPs were IMP3-immunoprecipitated from the respective peak fractions (NFATC3, #15; ANKRD17, #17), using CAMSAP1 circRNA (peak fraction #11) and anti-eIF4E immunoprecipitation as negative controls. Immunoprecipitation efficiencies (% of input; statistical deviations based on technical triplicates) were determined by RT-qPCR.

    Journal: Scientific Reports

    Article Title: CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    doi: 10.1038/srep31313

    Figure Lengend Snippet: IMP3 protein specifically and stably bound to circRNA family: validation and characterization. ( A ) Validation of IMP3 bound to specific circRNAs. Lysates of HepG2, PANC1, and PATU cells were subjected to immunoprecipitation with anti-IMP3 antibodies (IP), or as mock control, with anti-FLAG antibodies (m). Co-precipitated RNA was purified and assayed by RT-PCR for FTL mRNA (positive control), CAMSAP1 (negative control circRNA), and for the putative IMP3-associated circRNAs CDYL, NFATC3, and ANKRD17. For each of the circRNAs, the linear isoform of the respective gene was tested in addition (circ/lin; 90% of the mock- and IMP3-immunoprecipitates were used in RT-PCR). In addition, 5% of the input material was assayed (I). M, markers (in bp). ( B ) Quantitative immunoprecipitation analysis of IMP3-circRNA association in three cell lines (HepG2, PANC1, and PATU). For the same set of IMP3 circRNA targets and controls as shown in panel A, the immunoprecipitation efficiences were determined by RT-qPCR assays (% of input; statistical deviations based on biological duplicates). ( C ) Sedimentation profiles of IMP3-containing circRNPs. Cytoplasmic S100 extract from HeLa cells (extract), corresponding free RNA (RNA), and proteinase K-treated extract (extract + PK) were fractionated by glycerol gradient centrifugation (#1–22; the last fraction contains the resuspended pellet), followed by RT-PCR analysis of two IMP3-containing circRNAs (NFATC3 and ANKRD17). The relatively low recovery of circRNAs NFATC3 (1298 nt) and especially ANKRD17 (1832 nt), the largest circRNAs analyzed in S100 extract, may be caused by the higher tendency of such large circRNPs to aggregate and form precipitates, which were lost in the pellet fraction. The positions of ribosomal RNA size markers are indicated (5S, 18S, and 28S), as well as the shift of the circRNA vs. circRNP peak fractions (brackets). For comparison, the distribution of total IMP3 protein across the gradient was visualized by Western blotting in extract and, as a control, in proteinase K-treated extract. ( D ) IMP3 immunoprecipitation efficiencies of gradient-purified NFATC3 and ANKRD17 circRNPs. HeLa cytoplasmic S100 extract was gradient-fractionated, and NFATC3 and ANKRD17 circRNPs were IMP3-immunoprecipitated from the respective peak fractions (NFATC3, #15; ANKRD17, #17), using CAMSAP1 circRNA (peak fraction #11) and anti-eIF4E immunoprecipitation as negative controls. Immunoprecipitation efficiencies (% of input; statistical deviations based on technical triplicates) were determined by RT-qPCR.

    Article Snippet: After a 20 min incubation at room temperature, the samples were washed three times with 1 ml of washing buffer (10 mM Tris-HCl pH 7.5, 100/300/600 mM KCl, 2.5 mM MgCl2 , 0.1% Triton X-100), treated with proteinase K (Roth), phenol/chloroform extracted, and ethanol precipitated.

    Techniques: Stable Transfection, Immunoprecipitation, Purification, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Quantitative RT-PCR, Sedimentation, Gradient Centrifugation, Western Blot