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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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    micrococcal nuclease - by Bioz Stars, 2020-09
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    99
    Thermo Fisher rna
    MS2-driven <t>RNA</t> packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of <t>DNAse-treated</t> supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin
    MS2-driven <t>RNA</t> packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of <t>DNAse-treated</t> supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
    Trypsin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 24572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore collagenase
    MS2-driven <t>RNA</t> packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of <t>DNAse-treated</t> supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
    Collagenase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20723 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mgcl2
    MS2-driven <t>RNA</t> packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of <t>DNAse-treated</t> supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
    Mgcl2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mgcl2
    MS2-driven <t>RNA</t> packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of <t>DNAse-treated</t> supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.
    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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    Millipore bsa
    Subcellular localization of TL-binding sites in T cruzi by transmission electron microscopy (TEM). Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of <t>BSA-gold</t> as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [ 42 ]). Cryosections were sequentially probed with <t>biotinylated</t> TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 42788 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trypsin
    Subcellular localization of TL-binding sites in T cruzi by transmission electron microscopy (TEM). Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of <t>BSA-gold</t> as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [ 42 ]). Cryosections were sequentially probed with <t>biotinylated</t> TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.
    Trypsin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore triton x 100
    Surface localization and effect on vesicle protein composition of Hbp (chimeras) in  E. coli  OMVs. (A and B) Equal amounts of intact OMVs (−tx) or OMVs permeabilized with Triton X-100 (+tx) from  E. coli  JC8031 harboring the empty vector (EV) or
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher edta
    Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% <t>agarose-Tris</t> acetate <t>EDTA</t> gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.
    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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    Millipore hepes
    Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% <t>agarose-Tris</t> acetate <t>EDTA</t> gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.
    Hepes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 50050 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega trypsin
    Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% <t>agarose-Tris</t> acetate <t>EDTA</t> gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.
    Trypsin, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 39092 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnase i
    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or <t>DNase</t> I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p
    Dnase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i
    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
    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 pbs
    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 45702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore edta
    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
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    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
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    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
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    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with <t>DNase</t> I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P
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    Partial characterization of DSB repair complexes by co-IP analysis. NEs <t>(benzonase-treated)</t> from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with ( a ) anti-RNAP II (pSer2, H5 Ab); ( b ) anti-53BP1; ( c ) anti-Lig IV; ( d ) anti-PNKP; ( e ) anti-PARP1; or ( f ) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.
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    Partial characterization of DSB repair complexes by co-IP analysis. NEs <t>(benzonase-treated)</t> from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with ( a ) anti-RNAP II (pSer2, H5 Ab); ( b ) anti-53BP1; ( c ) anti-Lig IV; ( d ) anti-PNKP; ( e ) anti-PARP1; or ( f ) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.
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    Partial characterization of DSB repair complexes by co-IP analysis. NEs <t>(benzonase-treated)</t> from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with ( a ) anti-RNAP II (pSer2, H5 Ab); ( b ) anti-53BP1; ( c ) anti-Lig IV; ( d ) anti-PNKP; ( e ) anti-PARP1; or ( f ) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.
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    Partial characterization of DSB repair complexes by co-IP analysis. NEs <t>(benzonase-treated)</t> from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with ( a ) anti-RNAP II (pSer2, H5 Ab); ( b ) anti-53BP1; ( c ) anti-Lig IV; ( d ) anti-PNKP; ( e ) anti-PARP1; or ( f ) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.
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    Image Search Results


    MS2-driven RNA packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of DNAse-treated supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

    doi: 10.1038/mtm.2015.39

    Figure Lengend Snippet: MS2-driven RNA packaging into chimeric MS2-coat lentiviral particles. ( a ) Vectors for mRNA recruitment into MS2RLP. RNA transfer vector: control and reporter constructs. plenti_Luc: lentiviral vector serving as positive control for Ψ-dependent packaging. pLuc: a pcDNA-derived vector expressing the luciferase gene. Luc_MS2_12X: model construct designed to evaluate MS2-Coat protein based recruitment, the luciferase gene expressed under the CMV promoter contains 12 copies of the 19-nt RNA stem-loop from MS2 bacteriophage (MS2_12X). BGH-pA: Bovine growth hormone poly-adenylation signal. In red, detail of the sequence for two repeats. Packaging sequence: p8.74 (WT): standard HIV-1 trans-complementation, p8.74 ΔZF (ΔZF): derived from WT by deletion of the ZF2 in Nucleocapsid (NC). p8.74 ΔZF-MS2coat (ΔZF_MS2coat): vector containing MS2-Coat protein in place of the ZF2. CA, capsid; MA, matrix; MS2-coat, coat protein from MS2 bacteriophage; NC, nucleocapsid; POL, polymerase. ( b ) Transmission electron microscopy. (1) Control integrative lentiviral vectors; (2) MS2RLPs, picture include the smaller and larger particles. Both particles were observed by transmission electron microscopy in negative staining conditions using a LaB6 JEOL JEM2100 electron microscope (magnification: ×52,800). Bars 50 nm. (3) Boxplot, distribution of the average size of particles measured from 52 pictures of integrative-lentiviral (ILV) (dark blue) and 35 of MS2RLP (light blue). Images and counts were obtained from a single production of each vector. ( c ) RT-qPCR analysis of luciferase mRNA level in MS2RLPs. Control and MS2RLP samples of DNAse-treated supernatant collected 48 hours after transfection. In all particles collected, no DNA amplification was observed without an RT step (data not shown). The analyzed volume was defined after normalization to CAp24 concentration in the supernatant. Data are mean ± SD ( n = 3, in duplicate). Gag-WT: lentiviral vector expressing a wild-type Gag. Gag-ΔZF: lentiviral vector expressing a ZF2 deleted Gag. Gag-ΔZF_MS2Coat: lentiviral vector expressing Gag with ZF2 replaced by MS2coat sequence. Env-Ampho: amphotropic envelope. pLenti_Luc: lentiviral vector expressing the Luciferase gene. Luc_MS2_12X: mRNA containing 12 repetitions of MS2 RNA stem loop and the Luciferase coding sequence. Combination of Gag-ΔZF_MS2Coat, Luc_MS2_12X.

    Article Snippet: In total, 4 µl RNA treated with TURBO DNAse (2 U/µl) was used for cDNA synthesis with the Superscript First-strand Synthesis System (Life Technologies).

    Techniques: Plasmid Preparation, Construct, Positive Control, Derivative Assay, Expressing, Luciferase, Sequencing, Transmission Assay, Electron Microscopy, Negative Staining, Microscopy, Quantitative RT-PCR, Transfection, Amplification, Concentration Assay

    Subcellular localization of TL-binding sites in T cruzi by transmission electron microscopy (TEM). Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of BSA-gold as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [ 42 ]). Cryosections were sequentially probed with biotinylated TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.

    Journal: PLoS ONE

    Article Title: Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

    doi: 10.1371/journal.pone.0163302

    Figure Lengend Snippet: Subcellular localization of TL-binding sites in T cruzi by transmission electron microscopy (TEM). Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of BSA-gold as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [ 42 ]). Cryosections were sequentially probed with biotinylated TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.

    Article Snippet: Parasites were labeled with either biotinylated TL (1% BSA in TBS, 1 mM CaCl2, 40 μg/ml biotinylated-TL (Sigma)) or biotinylated ricin (Sigma) and then revealed by streptavidin conjugated to Alexa 488 or Alexa 594 (Molecular Probes).

    Techniques: Binding Assay, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Incubation

    Surface localization and effect on vesicle protein composition of Hbp (chimeras) in  E. coli  OMVs. (A and B) Equal amounts of intact OMVs (−tx) or OMVs permeabilized with Triton X-100 (+tx) from  E. coli  JC8031 harboring the empty vector (EV) or

    Journal: Applied and Environmental Microbiology

    Article Title: Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

    doi: 10.1128/AEM.01941-14

    Figure Lengend Snippet: Surface localization and effect on vesicle protein composition of Hbp (chimeras) in E. coli OMVs. (A and B) Equal amounts of intact OMVs (−tx) or OMVs permeabilized with Triton X-100 (+tx) from E. coli JC8031 harboring the empty vector (EV) or

    Article Snippet: When required, OMVs were lysed by incubation with 0.5% (vol/vol) Triton X-100 (Sigma-Aldrich) for 15 min on ice.

    Techniques: Plasmid Preparation

    Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% agarose-Tris acetate EDTA gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65 ▿

    doi: 10.1128/JVI.00497-07

    Figure Lengend Snippet: Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% agarose-Tris acetate EDTA gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.

    Article Snippet: The resin was then washed with 10 ml of TEV buffer (150 mM NaCl, 25 mM Tris-Cl [pH 8.0], 3 mM MgCl2 , 0.5 mM EDTA, 1 mM DTT, 0.1% NP-40) and incubated overnight in 1 ml of TEV buffer containing 100 U of AcTEV protease (Invitrogen).

    Techniques: BAC Assay, Clone Assay, Purification, Labeling, Binding Assay, Marker

    Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Journal: Nature

    Article Title: Induced ncRNAs Allosterically Modify RNA Binding Proteins in cis to Inhibit Transcription

    doi: 10.1038/nature06992

    Figure Lengend Snippet: Consensus GGUG-containing RNA oligonucleotide promotes the inhibitory effect of TLS on CBP/p300 HAT activities a , Co-immunoprecipitation (IP) of p300 and TLS from RNase A-treated HeLa cells. b, P300 HAT activity was measured using micrococcal nuclease (MNase) or DNase I pre-treated GST and GST-TLS in the presence of GGUG- or CCUC-oligonucleotide. * p

    Article Snippet: RNase A, micrococcal nuclease (MNase), DNase I, RNase H and RNase T1 treatment Whole cell extracts of GST proteins were treated with RNase A (25 μg/50 μl, Sigma), and incubated on ice for 20 min. GST-TLS in whole cell extracts was sequentially treated with 10 μg of micrococcal nuclease (Roche) in 100 mM sodium glycine (pH 8.6) and 10 mM CaCl2 at 37 °C for 4 min, 0 °C for 1 min, and room temperature for 20 min, and terminated by addition of 10 mM EGTA, followed with or without incubation of 100 pmol/20 μl of RNA oligonucleotides.

    Techniques: HAT Assay, Immunoprecipitation, Activity Assay

    Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with DNase I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P

    Journal: Nucleic Acids Research

    Article Title: WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway

    doi: 10.1093/nar/gkq1338

    Figure Lengend Snippet: Roles of WDHD1 in the expression of centromeric repeat non-coding RNA. ( A ) Expression levels of centromeric non-coding RNA spanning major and minor satellite repeats was examined by RT–PCR analysis of RNA isolated from control (ctrl) or WDHD1 knockdown NIH-3T3 cells. Total RNA samples were treated with DNase I prior to reverse transcription. ‘−’ denotes RT-minus reactions in which no reverse transcriptase was added. Expression levels of the housekeeping gene GAPDH , and WDHD1 are also shown. ( B ) Effect of WDHD1 knockdown on the transcription rates of minor and major satellite repeat region, as indicated. Nuclear run-on assays were performed to monitor newly transcribed centromeric RNA from nuclei of control (ctrl) and WDHD1 knockdown NIH-3T3 cells. U5 snRNA, which remained unchanged in both cell types, was used to demonstrate uniformity of input RNA. ‘–’ denotes RT-minus reactions in which no reverse transcriptase was added. Quantitative results are shown by bar graph below, and represent the mean ± SD of three independent experiments. For statistical significance of quantitative comparisons, calculations were done by (* P

    Article Snippet: The nuclear run-on RNA and total RNA were then digested with DNase I (Ambion) to further remove contaminating genomic DNA.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation

    Partial characterization of DSB repair complexes by co-IP analysis. NEs (benzonase-treated) from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with ( a ) anti-RNAP II (pSer2, H5 Ab); ( b ) anti-53BP1; ( c ) anti-Lig IV; ( d ) anti-PNKP; ( e ) anti-PARP1; or ( f ) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.

    Journal: Nature Communications

    Article Title: Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    doi: 10.1038/ncomms13049

    Figure Lengend Snippet: Partial characterization of DSB repair complexes by co-IP analysis. NEs (benzonase-treated) from HEK293 cells (either mock (−) or Bleo- (+) treated, or allowed to recover (+/R, 10–12 h) after Bleo treatment) were immunoprecipitated (IP'd) with ( a ) anti-RNAP II (pSer2, H5 Ab); ( b ) anti-53BP1; ( c ) anti-Lig IV; ( d ) anti-PNKP; ( e ) anti-PARP1; or ( f ) anti-Lig IIIα antibodies (Abs, even lanes) or control IgG (odd lanes) and tested for the presence of associated proteins with specific Abs as indicated to the right of each row. Individual IP experiments were repeated at least three times from separate batches of cells, and one representative figure is shown in each case.

    Article Snippet: Supernatants were collected after centrifugation at 15,000g for 30 min and DNA/RNA in the suspension was digested with 0.15 U μl−1 benzonase (Novagen) at 37 °C for 1 h. The samples were centrifuged at 20,000g for 30 min, and the supernatants collected as NEs and stored in aliquots at −80 °C.

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation