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  • 99
    New England Biolabs micrococcal nuclease
    Micrococcal Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher subcellular protein fractionation kit
    Identification of ER membrane <t>protein</t> interactomes by proximity proteomics. (A) Schematic of known (SEC61 translocon, OST complex), and candidate (SEC62, LRRC59) ER-ribosome receptors. SEC61β (purple), a subunit of the SEC61 translocon, RPN1 (green), a subunit of the OST complex, SEC62 (orange), and LRRC59 (blue) are expressed as BioID chimeras, labeling interacting and near-neighbor proteins (indicated by starred ribosomes and proteins W, X, Y, and Z). (B) Left panel: Streptavidin blots examining the <t>subcellular</t> distribution of biotin-labeled proteins within HEK293 cells expressing either the LRRC59-, SEC62-, SEC61β-, or RPN1-BirA reporter constructs. Biotin labeling (doxycycline (dox)-inducible expression of reporters) was performed over a time course spanning 0-6 hours and cytosol (C) and membrane (M) extracts prepared by detergent <t>fractionation.</t> Right panel: Densitometric quantifications of biotin labeling intensities for cytosolic and membrane fractions. (C) Canine pancreas rough microsomes with (+BirA) or without (-BirA) the addition of BirA* in trans . Biotin labeling of proteins was conducted over 0-18 hours (top, left). Biotin labeling intensities were quantified using densitometric analyses (top, right). As a loading control, total protein lysate was analyzed by India ink staining (bottom, left) and quantified by densitometric analysis (bottom, right).
    Subcellular Protein Fractionation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher micrococcal nuclease
    Identification of ER membrane <t>protein</t> interactomes by proximity proteomics. (A) Schematic of known (SEC61 translocon, OST complex), and candidate (SEC62, LRRC59) ER-ribosome receptors. SEC61β (purple), a subunit of the SEC61 translocon, RPN1 (green), a subunit of the OST complex, SEC62 (orange), and LRRC59 (blue) are expressed as BioID chimeras, labeling interacting and near-neighbor proteins (indicated by starred ribosomes and proteins W, X, Y, and Z). (B) Left panel: Streptavidin blots examining the <t>subcellular</t> distribution of biotin-labeled proteins within HEK293 cells expressing either the LRRC59-, SEC62-, SEC61β-, or RPN1-BirA reporter constructs. Biotin labeling (doxycycline (dox)-inducible expression of reporters) was performed over a time course spanning 0-6 hours and cytosol (C) and membrane (M) extracts prepared by detergent <t>fractionation.</t> Right panel: Densitometric quantifications of biotin labeling intensities for cytosolic and membrane fractions. (C) Canine pancreas rough microsomes with (+BirA) or without (-BirA) the addition of BirA* in trans . Biotin labeling of proteins was conducted over 0-18 hours (top, left). Biotin labeling intensities were quantified using densitometric analyses (top, right). As a loading control, total protein lysate was analyzed by India ink staining (bottom, left) and quantified by densitometric analysis (bottom, right).
    Micrococcal Nuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cell lysis
    Identification of ER membrane <t>protein</t> interactomes by proximity proteomics. (A) Schematic of known (SEC61 translocon, OST complex), and candidate (SEC62, LRRC59) ER-ribosome receptors. SEC61β (purple), a subunit of the SEC61 translocon, RPN1 (green), a subunit of the OST complex, SEC62 (orange), and LRRC59 (blue) are expressed as BioID chimeras, labeling interacting and near-neighbor proteins (indicated by starred ribosomes and proteins W, X, Y, and Z). (B) Left panel: Streptavidin blots examining the <t>subcellular</t> distribution of biotin-labeled proteins within HEK293 cells expressing either the LRRC59-, SEC62-, SEC61β-, or RPN1-BirA reporter constructs. Biotin labeling (doxycycline (dox)-inducible expression of reporters) was performed over a time course spanning 0-6 hours and cytosol (C) and membrane (M) extracts prepared by detergent <t>fractionation.</t> Right panel: Densitometric quantifications of biotin labeling intensities for cytosolic and membrane fractions. (C) Canine pancreas rough microsomes with (+BirA) or without (-BirA) the addition of BirA* in trans . Biotin labeling of proteins was conducted over 0-18 hours (top, left). Biotin labeling intensities were quantified using densitometric analyses (top, right). As a loading control, total protein lysate was analyzed by India ink staining (bottom, left) and quantified by densitometric analysis (bottom, right).
    Cell Lysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4395 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher mnase
    Digestion of different regions of the rDNA units with <t>MNase.</t> (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at <t>24°C</t> for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
    Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher magnetic chip kit
    Digestion of different regions of the rDNA units with <t>MNase.</t> (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at <t>24°C</t> for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was
    Magnetic Chip Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase a
    Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3  fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3  pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3  further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3  in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and  Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.
    Rnase A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher agarose chip kit
    Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3  fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3  pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3  further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3  in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and  Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.
    Agarose Chip Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher proteinase k
    Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3  fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3  pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3  further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3  in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and  Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.
    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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    Thermo Fisher edta
    Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3  fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3  pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3  further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3  in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and  Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.
    Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protein g dynabeads
    Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3  fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3  pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3  further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3  in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and  Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.
    Protein G Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher rnasealert lab test kit
    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with <t>RNaseAlert®</t> ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)
    Rnasealert Lab Test Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mgcl2
    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with <t>RNaseAlert®</t> ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)
    Mgcl2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 104037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nacl
    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with <t>RNaseAlert®</t> ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)
    Nacl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i
    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with <t>RNaseAlert®</t> ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 73125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sytox green
    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with <t>RNaseAlert®</t> ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)
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    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with <t>RNaseAlert®</t> ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)
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    Characterization of HBV DNA and <t>RNA</t> in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by <t>TRIzol</t> reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.
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    Image Search Results


    Identification of ER membrane protein interactomes by proximity proteomics. (A) Schematic of known (SEC61 translocon, OST complex), and candidate (SEC62, LRRC59) ER-ribosome receptors. SEC61β (purple), a subunit of the SEC61 translocon, RPN1 (green), a subunit of the OST complex, SEC62 (orange), and LRRC59 (blue) are expressed as BioID chimeras, labeling interacting and near-neighbor proteins (indicated by starred ribosomes and proteins W, X, Y, and Z). (B) Left panel: Streptavidin blots examining the subcellular distribution of biotin-labeled proteins within HEK293 cells expressing either the LRRC59-, SEC62-, SEC61β-, or RPN1-BirA reporter constructs. Biotin labeling (doxycycline (dox)-inducible expression of reporters) was performed over a time course spanning 0-6 hours and cytosol (C) and membrane (M) extracts prepared by detergent fractionation. Right panel: Densitometric quantifications of biotin labeling intensities for cytosolic and membrane fractions. (C) Canine pancreas rough microsomes with (+BirA) or without (-BirA) the addition of BirA* in trans . Biotin labeling of proteins was conducted over 0-18 hours (top, left). Biotin labeling intensities were quantified using densitometric analyses (top, right). As a loading control, total protein lysate was analyzed by India ink staining (bottom, left) and quantified by densitometric analysis (bottom, right).

    Journal: bioRxiv

    Article Title: Quantitative proteomics links the LRRC59 interactome to mRNA translation on the ER membrane

    doi: 10.1101/2020.03.04.975474

    Figure Lengend Snippet: Identification of ER membrane protein interactomes by proximity proteomics. (A) Schematic of known (SEC61 translocon, OST complex), and candidate (SEC62, LRRC59) ER-ribosome receptors. SEC61β (purple), a subunit of the SEC61 translocon, RPN1 (green), a subunit of the OST complex, SEC62 (orange), and LRRC59 (blue) are expressed as BioID chimeras, labeling interacting and near-neighbor proteins (indicated by starred ribosomes and proteins W, X, Y, and Z). (B) Left panel: Streptavidin blots examining the subcellular distribution of biotin-labeled proteins within HEK293 cells expressing either the LRRC59-, SEC62-, SEC61β-, or RPN1-BirA reporter constructs. Biotin labeling (doxycycline (dox)-inducible expression of reporters) was performed over a time course spanning 0-6 hours and cytosol (C) and membrane (M) extracts prepared by detergent fractionation. Right panel: Densitometric quantifications of biotin labeling intensities for cytosolic and membrane fractions. (C) Canine pancreas rough microsomes with (+BirA) or without (-BirA) the addition of BirA* in trans . Biotin labeling of proteins was conducted over 0-18 hours (top, left). Biotin labeling intensities were quantified using densitometric analyses (top, right). As a loading control, total protein lysate was analyzed by India ink staining (bottom, left) and quantified by densitometric analysis (bottom, right).

    Article Snippet: Subcellular fractions were cleared by centrifugation (15,300 x g for 10 minutes).

    Techniques: Labeling, Expressing, Construct, Fractionation, Staining

    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: Subcellular fractionation of cells For subcellular fractionation of NOCT (1-431)-3F HEK293T cells, mitochondrial isolation was performed as previously described, with the omission of the sucrose gradient step ( ).

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

    Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

    Journal: Molecular and Cellular Biology

    Article Title: Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster ▿

    doi: 10.1128/MCB.01409-06

    Figure Lengend Snippet: Digestion of different regions of the rDNA units with MNase. (A) Isolated 1- to 16-h embryonic nuclei were digested with MNase (0.5 U/μl) at 24°C for 0, 1, or 4 min (lanes 2 to 4 of each panel). Purified (protein-free) genomic DNA was

    Article Snippet: For micrococcal nuclease (MNase) digestion, the nucleus solutions were made 3 mM CaCl2 and 0.5 units/μl MNase (Fermentas) and incubated at 24°C for the specified time.

    Techniques: Isolation, Purification

    Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3  fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3  pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3  further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3  in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and  Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.

    Journal: Nucleic Acids Research

    Article Title: Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

    doi: 10.1093/nar/gkt988

    Figure Lengend Snippet: Twinkle is membrane associated. ( A ) Isolated mitochondria of HEK293E cells were subjected to either KCl or sodium carbonate extraction (Na 2 CO 3 ) as described in the main text. Endogenous Twinkle was detected using a monoclonal antibody and blots were re-probed with antibodies for TFAM and mtSSB. Results show that endogenous Twinkle fractionates mostly to the pellet fraction using both methods illustrating its strong membrane association, similar to overexpressed Twinkle ( Supplementary Figure S6C ), whereas TFAM and mtSSB mostly became soluble, in particular, in combination with 0.5 M KCl and sonication. Please note that to detect Twinkle with confidence, more protein was used for these western blots sometimes resulting in overloading of TFAM. Sonication in combination with DNAseI (D) or DNaseI/RNase A (R, RNase)/Benzonase ( B ) released more TFAM and mtSSB than sonication alone, showing that a proportion of TFAM and mtSSB can be found in the insoluble fractions of the various experiments by means of their interaction with mtDNA (rightmost blot panel A). (B) Na 2 CO 3 fractionation shows that overexpressed Twinkle–Myc is almost exclusively in the pellet (p) fraction again indicative of tight membrane association, whereas TFAM is partially soluble (s = supernatant) (left panel). Treatment of the Na 2 CO 3 pellet fraction by DNAseI and subsequent re-extraction using Na 2 CO 3 further released a proportion of TFAM (middle panel). Finally, Na 2 CO 3 in combination with Triton-X100 (TX100) treatment solubilized both Twinkle–Myc and endogenous TFAM (right panel). ( C ) Similar to results shown in panel A, sonication in combination with DNAseI ( D ) or DNaseI/RNase A (R, RNase)/Benzonase (B) released more TFAM than sonication alone or combined with RNase treatment, showing that a proportion of TFAM can be found in the insoluble fractions of the various experiments by means of its interaction with mtDNA, whereas in this case, overexpressed Twinkle–Myc remains in the pellet fraction with all treatments. (D) Digitonin-based isolation of a mitochondrial membrane fraction again showed the retention of Twinkle and proportions of nucleoid-associated proteins such as TFAM, mtSSB and POLG1 (see main text and Supplementary Figure S6D ). This membrane fraction (marked as ‘Digitonin pellet’) was here subjected to flotation by layering a gradient of iodixanol on top of this fraction that was first re-solubilized with 1% TX100 (see ‘Materials and Methods’ for details). The gradient was then subjected to ultracentrifugation. In parallel, a mitochondrial fraction that was directly solubilized by 1% TX100 (marked as ‘TX100’) was subjected to the same procedure. Collected fractions were isolated as indicated and subjected to western blot analysis as well as dot blot analysis to detect mtDNA. The results show that mtDNA and nucleoid-associated proteins in the digitonin-lysis membrane fraction moved up the gradient to a single low-density iodixanol concentration, showing that they likely form a single complex. In contrast, COXII and a marker for the large ribosomal subunit MRPL49 have remained at relatively high-density fractions. In the total mitochondrial TX100 lysate, Twinkle and other nucleoid proteins appear more dispersed as does mtDNA.

    Article Snippet: Treatment of isolated mitochondria by sonication and nucleases Mitochondria were resuspended in enzyme-buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 3 mM CaCl2 , 2 mM MgCl2 ), sonicated on ice at 40% power for three times 20 s before addition of the enzymes as indicated DNase I (Fermentas) 10U, RNAse A (Fermentas) 20 µg, Micrococcal nuclease (Fermentas) 50 U and Benzonase nuclease (Sigma) 50U, and incubated at +37°C for 30 min. Where appropriate, lysates were further subjected to carbonate extraction as described above.

    Techniques: Isolation, Sonication, Western Blot, Fractionation, Dot Blot, Lysis, Concentration Assay, Marker

    SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with RNaseAlert® ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)

    Journal: Nature Communications

    Article Title: Cell entry of a host-targeting protein of oomycetes requires gp96

    doi: 10.1038/s41467-018-04796-3

    Figure Lengend Snippet: SpHtp3 is a self-translocating nuclease. a Amino acid sequence of SpHtp3 (top), including the secretion signal (M1-G21, underlined), the RxLR sequence (R48-R51, red) and the predicted nuclease domain (L89-S197, bold). Protein domain structure of SpHtp3 (bottom). b Visualisation of RNA (left, RTG-2 cell RNA) and DNA (right, linearised pET21b) degrading activities of SpHtp3-His 6 and SpHtp3-mRFP ( n = 3). c Real-time ribonuclease activity assessment of SpHtp3 wt (black) compared to a negative control (SpHtp1-mRFP, red) and a non-functional mutant of SpHtp3 (GTLG, blue) with RNaseAlert® ( n = 2). d Autonomous translocation activity of recombinant SpHtp3-mRFP into living RTG-2 cells at pH 7.5 and 5.5. The control (mRFP only) does not show any translocation. Scale bar: 20 µm ( n = 3)

    Article Snippet: Real-time fluorescent monitoring of ribonuclease activity was performed at RT using the RNaseAlert(R) Lab Test Kit (#AM1964, Applied Biosystems).

    Techniques: Sequencing, Activity Assay, Negative Control, Functional Assay, Mutagenesis, Translocation Assay, Recombinant

    Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Southern Blot, Incubation, Labeling, Northern Blot, Electrophoresis

    Mapping and identifying 3′ ends of extracellular HBV RNAs. (A) Northern blot detection of extracellular HBV RNAs with various riboprobes. Viral RNA from cytoplasmic (C) nucleocapsids (lanes 2, 5, 8, 11, 14, and 17) or culture supernatant (S) (lanes 3, 6, 9, 12, 15, and 18) of HepAD38 cells was extracted with TRIzol reagent and treated with DNase I before Northern blot analysis with plus-strand-specific riboprobes spanning the HBV genome as indicated. pgRNA was used as a reference, and map coordinates were numbered according to the sequence of the HBV genome (genotype D, accession number AJ344117.1 ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Mapping and identifying 3′ ends of extracellular HBV RNAs. (A) Northern blot detection of extracellular HBV RNAs with various riboprobes. Viral RNA from cytoplasmic (C) nucleocapsids (lanes 2, 5, 8, 11, 14, and 17) or culture supernatant (S) (lanes 3, 6, 9, 12, 15, and 18) of HepAD38 cells was extracted with TRIzol reagent and treated with DNase I before Northern blot analysis with plus-strand-specific riboprobes spanning the HBV genome as indicated. pgRNA was used as a reference, and map coordinates were numbered according to the sequence of the HBV genome (genotype D, accession number AJ344117.1 ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Northern Blot, Sequencing, Clone Assay