biotinylated pcr Search Results


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  • 93
    Thermo Fisher biotinylated
    Chordin affinity columns bind <t>biotinylated</t> cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.
    Biotinylated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore biotinylated isoxazole
    Acutely active E 2 -responsive MegaTrans enhancers concentrate a protein complex that can undergo phase transition. a , Schematic representation of the ERα/MegaTrans complex recruited to E 2 -activated enhancers, which transcribe eRNAs and recruit the condensin I/II complexes. b , Western blot analyses showing that ERα, several MegaTrans components and condensin component SMC4 are precipitated by <t>biotinylated</t> isoxazole (b-isoxazole). FUS and GAPDH proteins are used as a positive and negative control for the assay, respectively. WCL, whole-cell lysate. c,d , FRAP data on phase-separated droplet formed in vitro by purified recombinant GATA3-maltose binding protein (MBP) ( c ) and ERα-MBP ( d ). Top, charts show individual data points represented by dots, lines represent fitting to an exponential model to estimate the half-time of recovery. Bottom, representative images of in vitro droplets before and after photobleaching. e , Fluorescence microscopy images of a representative nucleus from MCF7 cells transfected with ERα-mCherry, before (−E 2 ) or after (+E 2 , 5 or 15min) E 2 treatment. Scale bar, 2 μm. f , Mean intensity and photobleaching normalized fluorescence of ERα-mTurquoise foci in E 2 treated MCF7 cells relative to pre-bleaching signal. Error bars represent s.e.m. of n ≥10 cells per time point. g , Levels of eRNA from indicated enhancers, measured by reverse transcription PCR, from MCF7 cells depleted of endogenous GATA3 and expressing either wild type or IDR-deleted GATA3 (GATA3-IDR mut), after 1 h E 2 stimulation. shCTL indicates non-targeting control shRNA. The IDR (aa 2–250) is shown in the schematics on top. Results are shown as individual data points (circles), mean±s.d. (lines). P values were calculated with an unpaired Student’s t -test. Data are representative of three independent experiments.
    Biotinylated Isoxazole, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche avidin biotin immunoperoxidase reaction
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Avidin Biotin Immunoperoxidase Reaction, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche biotinylated pcr
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Biotinylated Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    EpigenDx biotinylated pcr primers
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Biotinylated Pcr Primers, supplied by EpigenDx, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher biotinylated pcr amplicon
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Biotinylated Pcr Amplicon, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    EpigenDx single stranded biotinylated pcr
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Single Stranded Biotinylated Pcr, supplied by EpigenDx, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Diatech Pharmacogenetics biotinylated single stranded pcr
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Biotinylated Single Stranded Pcr, supplied by Diatech Pharmacogenetics, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneWorks biotinylated
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Biotinylated, supplied by GeneWorks, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Roche biotinylated primer
    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint <t>immunoperoxidase</t> reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).
    Biotinylated Primer, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher biotinylated primers
    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for <t>biotinylated</t> DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Biotinylated Primers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Eurofins biotinylated primers
    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for <t>biotinylated</t> DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Biotinylated Primers, supplied by Eurofins, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher biotinylated dutp
    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for <t>biotinylated</t> DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Biotinylated Dutp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche biotinylated dutp
    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for <t>biotinylated</t> DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Biotinylated Dutp, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche biotinylated ntps
    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for <t>biotinylated</t> DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Biotinylated Ntps, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore biotinylated primers
    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for <t>biotinylated</t> DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Biotinylated Primers, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore biotinylated aptamers
    eIF4e <t>aptamers</t> inhibit cell proliferation in HeLa and HEK293 cells. HeLa and HEK293 cells were transfected with varying concentrations of aptamers using Lipofectamine 2000 and 2 days after transfection, cell viability was measured with Cell Titer Blue for 3 hours. ( a ) HEK293 cells transfected with 100 nmol/l of each aptamer. NS is an oligonucleotide that can form hairpin structures. ( b ) HEK293 and ( c ) HeLa cells transfected with varying amount of aptamers. ( d ) HEK293 cells transfected with shRNAs against eIF4e or a control shRNA against DMPK TA cloned into pCR2.1. ( e ) HEK293 cells transfected with 100 nmol/l of each aptamer. ( f ) HEK293 cells were transfected with 100 nmol/l of <t>biotinylated</t> Apt 6 or biotinylated NS. Six hours after transfection, cells were fixed with paraformaldehyde and lysed. Cell lysates were then incubated with Streptavidin Dynabeads. Western blots of the input, unbound, and bound fractions were probed with an eIF4e antibody. ** P
    Biotinylated Aptamers, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher biotinylated utps
    eIF4e <t>aptamers</t> inhibit cell proliferation in HeLa and HEK293 cells. HeLa and HEK293 cells were transfected with varying concentrations of aptamers using Lipofectamine 2000 and 2 days after transfection, cell viability was measured with Cell Titer Blue for 3 hours. ( a ) HEK293 cells transfected with 100 nmol/l of each aptamer. NS is an oligonucleotide that can form hairpin structures. ( b ) HEK293 and ( c ) HeLa cells transfected with varying amount of aptamers. ( d ) HEK293 cells transfected with shRNAs against eIF4e or a control shRNA against DMPK TA cloned into pCR2.1. ( e ) HEK293 cells transfected with 100 nmol/l of each aptamer. ( f ) HEK293 cells were transfected with 100 nmol/l of <t>biotinylated</t> Apt 6 or biotinylated NS. Six hours after transfection, cells were fixed with paraformaldehyde and lysed. Cell lysates were then incubated with Streptavidin Dynabeads. Western blots of the input, unbound, and bound fractions were probed with an eIF4e antibody. ** P
    Biotinylated Utps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenePharma Company biotinylated nc
    The underlying mechanism of the regulation of miR-138 and LINC00152. ( a ) Amount of LINC00152 bound to SNRNP70 (a positive control), Ago2 or IgG (a negative control) was measured by qPCR after RIP in NOZ cells. ( b ) NOZ cells were transfected with <t>biotinylated</t> NC (Bio-NC), biotinylated wild-type miR-138 (Bio-miR-138) or biotinylated mutant miR-138 (Bio-miR-138-mut), and biotin-based miRNA pull-down assays were conducted after 48 h of transfection. LINC00152 levels were analysed by qPCR. ( c ) Luciferase reporter activity in NOZ cells was detected after co-transfection with pPG-miR-138 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type LINC00152 (pmiR-GLo-LINC00152-wt) or mutant transcripts (pmiR-GLo-LINC00152-mut). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. ( d ) Luciferase reporter activity in NOZ cells was detected after co-transfection with pPG-miR-138 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type HIF-1α (pmiR-GLo-HIF-1α-wt) or mutant transcripts (pmiR-GLo-HIF-1α-mut). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. The mean ± s.d. of triplicate experiments were plotted. ** p
    Biotinylated Nc, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Seikagaku biotinylated habp
    Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with <t>biotinylated</t> <t>HABP</t> and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).
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    Ribobio biotinylated nc
    Validation of TUG1 as a direct target of miR-138-5p (A) The sequences of the predicted miR-138-5p binding site and the TUG1 segments containing the wild type binding site are shown. (B) Dual-luciferase reporter assay revealed that miR-138-5p mimics decreased luciferase activity of pmirGLO-TUG1-Wt, but not of pmirGLO-TUG1-mut. (C) Detection of miR-138-5p using qRT-PCR in the sample pulled down by <t>biotinylated</t> TUG1 probe. (D) miR-138-5p expression in 30 paired of cervical cancer tissues was measured by qRT-PCR. (E) The correlation analysis was performed between TUG1 expression and miR-138-5p in cervical cancer tissues. *P
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    96
    Thermo Fisher neutravidin
    EGF detection using the immuno-phage assay in 50% BAL fluid VEGF (26 aM - 2.6 pM) was spiked into 50% BAL fluid (in PBS), and incubated with polyclonal anti-VEGF antibody functionalized magnetic particles. The biotinylated <t>antibody/Neutravidin/phage</t> affinity reagent was added, followed by washing. The sample was then analyzed using real-time PCR with phage-specific primers (n=6, error bars = ±1 SD, Non-template control C t ≥34). The No-VEGF control (Ct =22.67±0.55) is shown as a solid line with +/− one standard deviation represented by dotted lines.
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    Becton Dickinson biotinylated antibodies
    (A) PCs express BLyS receptor. Immune BM and spleen (SP) was isolated and stained with αCD138, αB220, and <t>biotinylated</t> BLyS. Histograms of BLyS receptor expression are shown for B220 + CD138 − cells (red); B220 − CD138 − cells (green); and B220 − CD138 + cells (blue). Data are representative of three independent experiments. (B) BM PCs express heightened levels of BCMA mRNA but not BAFF-R or TACI. RNA from splenic B cells (B cells), αCD40-induced B cell blasts (αCD40 B), or BM PCs (PC) was isolated via the TRIzol method. Real-time RT-PCR was performed with each sample to evaluate the relative levels of mRNA expression for BCMA, TACI, and BAFF-R. Data are representative of three experiments. (C) BLyS enhances Mcl-1 expression in PCs but not B cells. Purified CD138 + BM PCs or splenic B cells were cultured with the indicated stimulus, as described in Fig. 1 . After 18 h, RNA was isolated, and the relative expression of antiapoptotic genes was determined relative to β-actin.
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    Image Search Results


    Chordin affinity columns bind biotinylated cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.

    Journal: EMBO Reports

    Article Title: Integrin-?3 mediates binding of Chordin to the cell surface and promotes its endocytosis

    doi: 10.1038/sj.embor.embor902

    Figure Lengend Snippet: Chordin affinity columns bind biotinylated cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.

    Article Snippet: Cell-surface proteins from COS-7 cells were biotinylated (using EZ-link-Biotin; Pierce), solubilized with 0.1% Triton X-100 (as in ), and loaded on Chd–Fc or s-Fc affinity columns.

    Techniques: Immunoprecipitation, In Situ Hybridization, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Acutely active E 2 -responsive MegaTrans enhancers concentrate a protein complex that can undergo phase transition. a , Schematic representation of the ERα/MegaTrans complex recruited to E 2 -activated enhancers, which transcribe eRNAs and recruit the condensin I/II complexes. b , Western blot analyses showing that ERα, several MegaTrans components and condensin component SMC4 are precipitated by biotinylated isoxazole (b-isoxazole). FUS and GAPDH proteins are used as a positive and negative control for the assay, respectively. WCL, whole-cell lysate. c,d , FRAP data on phase-separated droplet formed in vitro by purified recombinant GATA3-maltose binding protein (MBP) ( c ) and ERα-MBP ( d ). Top, charts show individual data points represented by dots, lines represent fitting to an exponential model to estimate the half-time of recovery. Bottom, representative images of in vitro droplets before and after photobleaching. e , Fluorescence microscopy images of a representative nucleus from MCF7 cells transfected with ERα-mCherry, before (−E 2 ) or after (+E 2 , 5 or 15min) E 2 treatment. Scale bar, 2 μm. f , Mean intensity and photobleaching normalized fluorescence of ERα-mTurquoise foci in E 2 treated MCF7 cells relative to pre-bleaching signal. Error bars represent s.e.m. of n ≥10 cells per time point. g , Levels of eRNA from indicated enhancers, measured by reverse transcription PCR, from MCF7 cells depleted of endogenous GATA3 and expressing either wild type or IDR-deleted GATA3 (GATA3-IDR mut), after 1 h E 2 stimulation. shCTL indicates non-targeting control shRNA. The IDR (aa 2–250) is shown in the schematics on top. Results are shown as individual data points (circles), mean±s.d. (lines). P values were calculated with an unpaired Student’s t -test. Data are representative of three independent experiments.

    Journal: Nature structural & molecular biology

    Article Title: Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly

    doi: 10.1038/s41594-019-0190-5

    Figure Lengend Snippet: Acutely active E 2 -responsive MegaTrans enhancers concentrate a protein complex that can undergo phase transition. a , Schematic representation of the ERα/MegaTrans complex recruited to E 2 -activated enhancers, which transcribe eRNAs and recruit the condensin I/II complexes. b , Western blot analyses showing that ERα, several MegaTrans components and condensin component SMC4 are precipitated by biotinylated isoxazole (b-isoxazole). FUS and GAPDH proteins are used as a positive and negative control for the assay, respectively. WCL, whole-cell lysate. c,d , FRAP data on phase-separated droplet formed in vitro by purified recombinant GATA3-maltose binding protein (MBP) ( c ) and ERα-MBP ( d ). Top, charts show individual data points represented by dots, lines represent fitting to an exponential model to estimate the half-time of recovery. Bottom, representative images of in vitro droplets before and after photobleaching. e , Fluorescence microscopy images of a representative nucleus from MCF7 cells transfected with ERα-mCherry, before (−E 2 ) or after (+E 2 , 5 or 15min) E 2 treatment. Scale bar, 2 μm. f , Mean intensity and photobleaching normalized fluorescence of ERα-mTurquoise foci in E 2 treated MCF7 cells relative to pre-bleaching signal. Error bars represent s.e.m. of n ≥10 cells per time point. g , Levels of eRNA from indicated enhancers, measured by reverse transcription PCR, from MCF7 cells depleted of endogenous GATA3 and expressing either wild type or IDR-deleted GATA3 (GATA3-IDR mut), after 1 h E 2 stimulation. shCTL indicates non-targeting control shRNA. The IDR (aa 2–250) is shown in the schematics on top. Results are shown as individual data points (circles), mean±s.d. (lines). P values were calculated with an unpaired Student’s t -test. Data are representative of three independent experiments.

    Article Snippet: Protein supernatant was collected by centrifugation at 4 °C, 16,500g for 15 min. Then, 5% lysates were saved for whole cell extract control and remaining were aliquoted equally and incubated with various concentrations (10, 30 and 100 μM) biotinylated isoxazole (Sigma, catalog no. 900572) at 4 °C for 1 h with rotation.

    Techniques: Sublimation, Western Blot, Negative Control, In Vitro, Purification, Recombinant, Binding Assay, Fluorescence, Microscopy, Transfection, Polymerase Chain Reaction, Expressing, shRNA

    Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).

    Journal: Emerging Infectious Diseases

    Article Title: Bovine Leukemia Virus DNA in Human Breast Tissue

    doi: 10.3201/eid2005.131298

    Figure Lengend Snippet: Localization of bovine leukemia virus (BLV) in human breast tissue and bovine mammary epithelium samples detected by in situ PCR for the BLV tax region and immunohistochemical testing for p24 capsid protein. A) BLV-positive fetal lamb kidney (FLK) cell line. Brown at left indicates positive diaminobenzidine endpoint immunoperoxidase reaction to detect digoxygenin incorporated into PCR product within FLK cells. FLK cells reacted with PCR reaction mix without primers (right) to check for false-positive background show no reaction. Original magnification ×400. B) BLV-negative cell line Tb 1 Lu with (left) and without (right) primers. No reaction occurred with either condition because the cell line has no BLV to amplify and shows no nonspecific background. Original magnification ×400. C) BLV-positive lactating bovine mammary gland tissue with (left) and without (right) tax primers in the PCR mix. Dark brown at left indicates positive cells, some surrounding lumens filled with milk. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive due to nonspecific factors inherent in the tissue. Original magnification ×100. D) BLV-positive human tissue sample 010 reacted with tax primers. Dark brown at left indicates epithelial cells facing the lumen of a large cyst. Lack of reactive cells in sample at right without primers indicates reaction was not a false positive. Original magnification ×100. E) BLV-negative human tissue sample 143 exposed to PCR mix with (left) and without (right) primers showing no reaction with either condition in the epithelium of the long duct. Original magnification ×40. F) BLV-positive human tissue reacted with monoclonal antibody to BLV p24 (left) in an avidin-biotin-immunoperoxidase assay. Brown indicates end-point reaction in cytoplasm of epithelium projecting into the cyst lumen on a stalk of collagenous stroma. Note lack of reaction in sample at right with hybridoma medium substituted for primary antibody. Original magnification ×40. All cells and tissues were counterstained with Diff-Quik Solution II (Dade Behring, Newark, DE, USA).

    Article Snippet: Label incorporated into PCR products was detected by anti-dig antibodies in an avidin-biotin-immunoperoxidase reaction (Hoffman-La Roche).

    Techniques: In Situ, Polymerase Chain Reaction, Immunohistochemistry, Avidin-Biotin Assay, Diff-Quik

    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for biotinylated DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of

    Journal: Infection and Immunity

    Article Title: CcpA and LacD.1 Affect Temporal Regulation of Streptococcus pyogenes Virulence Genes ▿ Virulence Genes ▿ †

    doi: 10.1128/IAI.00746-09

    Figure Lengend Snippet: CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for biotinylated DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of

    Article Snippet: A biotinylated DNA fragment pull-down assay was adapted from the method of Grundling et al. ( ) as follows: biotinylated DNA fragments were generated by PCR using 5′ biotinylated primers (Invitrogen) (see Table S1 in the supplemental material) and HSC5 genomic DNA.

    Techniques: Generated, Polymerase Chain Reaction

    In vitro analysis of DREAM protein binding to cyclin B2 wild-type or mutant promoters. Nuclear extracts of density-arrested NIH3T3 cells were employed for DNA affinity purification using biotinylated human or mouse cyclin B2 promoters. ( A ) Binding was tested to wild-type, CDE, CHR or CDE/CHR mutant DNA probes by western blot analysis. As a negative control, a fragment of the Gapdhs promoter was used. As a protein binding to all cyclin B2 probes containing CCAAT-boxes, Nfya was detected. ( B ) Binding of the DREAM complex to the cyclin B2 promoters is independent of CCAAT-boxes. A truncated fragment of the mouse cyclin B2 promoter ending upstream of the CDE lacking the CCAAT-boxes was assayed for DREAM binding in comparison to a probe of the same length but with mutated CDE and CHR elements.

    Journal: Nucleic Acids Research

    Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

    doi: 10.1093/nar/gkr793

    Figure Lengend Snippet: In vitro analysis of DREAM protein binding to cyclin B2 wild-type or mutant promoters. Nuclear extracts of density-arrested NIH3T3 cells were employed for DNA affinity purification using biotinylated human or mouse cyclin B2 promoters. ( A ) Binding was tested to wild-type, CDE, CHR or CDE/CHR mutant DNA probes by western blot analysis. As a negative control, a fragment of the Gapdhs promoter was used. As a protein binding to all cyclin B2 probes containing CCAAT-boxes, Nfya was detected. ( B ) Binding of the DREAM complex to the cyclin B2 promoters is independent of CCAAT-boxes. A truncated fragment of the mouse cyclin B2 promoter ending upstream of the CDE lacking the CCAAT-boxes was assayed for DREAM binding in comparison to a probe of the same length but with mutated CDE and CHR elements.

    Article Snippet: DNA probes for affinity purification with the same sequence as hCCNB2-short and mCcnb2-short and truncated variants of mCcnb2-short without CCAAT-boxes (nt −130 to +1) were obtained by PCR using a biotinylated primer for labeling the 3′-end (Invitrogen, Carlsbad, CA, USA).

    Techniques: In Vitro, Protein Binding, Mutagenesis, Affinity Purification, Binding Assay, Western Blot, Negative Control

    The MMB complex binds and activates the cyclin B2 promoters through the CHR. ( A ) Nuclear extracts of proliferating NIH3T3 cells were employed for DNA affinity purification with biotinylated DNA probes of the human and mouse cyclin B2 promoters. Protein binding to wild-type, CDE, CHR or CDE/CHR mutant promoter fragments was tested by western blot analysis. As a protein binding to all cyclin B2 probes containing CCAAT-boxes, Nfya was detected. As a negative control, a fragment of the Gapdhs promoter was used. ( B ) Binding of the MMB complex to the mouse and human cyclin B2 DNA probes was assayed with DNA affinity purification of proteins derived from F9 cell nuclear extracts followed by western blot. Note that the DREAM complex components E2f4 and p130 do not bind to the probes. ( C ) To determine the effect of B-myb knockdown on the activity of mouse and human cyclin B2 promoters, NIH3T3 cells were transfected with the reporter constructs mouse Ccnb2 short and human CCNB2 short (wild-type or CHR mutant) together with vectors expressing shRNAs targeting B-myb (sh-Bmyb 1, sh-Bmyb 2). A shRNA construct expressing a non-targeting GFP-shRNA served as a negative control (sh-GFP). Luciferase activities were measured 48 h after transfection.

    Journal: Nucleic Acids Research

    Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

    doi: 10.1093/nar/gkr793

    Figure Lengend Snippet: The MMB complex binds and activates the cyclin B2 promoters through the CHR. ( A ) Nuclear extracts of proliferating NIH3T3 cells were employed for DNA affinity purification with biotinylated DNA probes of the human and mouse cyclin B2 promoters. Protein binding to wild-type, CDE, CHR or CDE/CHR mutant promoter fragments was tested by western blot analysis. As a protein binding to all cyclin B2 probes containing CCAAT-boxes, Nfya was detected. As a negative control, a fragment of the Gapdhs promoter was used. ( B ) Binding of the MMB complex to the mouse and human cyclin B2 DNA probes was assayed with DNA affinity purification of proteins derived from F9 cell nuclear extracts followed by western blot. Note that the DREAM complex components E2f4 and p130 do not bind to the probes. ( C ) To determine the effect of B-myb knockdown on the activity of mouse and human cyclin B2 promoters, NIH3T3 cells were transfected with the reporter constructs mouse Ccnb2 short and human CCNB2 short (wild-type or CHR mutant) together with vectors expressing shRNAs targeting B-myb (sh-Bmyb 1, sh-Bmyb 2). A shRNA construct expressing a non-targeting GFP-shRNA served as a negative control (sh-GFP). Luciferase activities were measured 48 h after transfection.

    Article Snippet: DNA probes for affinity purification with the same sequence as hCCNB2-short and mCcnb2-short and truncated variants of mCcnb2-short without CCAAT-boxes (nt −130 to +1) were obtained by PCR using a biotinylated primer for labeling the 3′-end (Invitrogen, Carlsbad, CA, USA).

    Techniques: Affinity Purification, Protein Binding, Mutagenesis, Western Blot, Negative Control, Binding Assay, Derivative Assay, Activity Assay, Transfection, Construct, Expressing, shRNA, Luciferase

    Strategy for the identification of proteins binding to the CDE/CHR tandem element of the mouse cyclin B2 promoter. NIH3T3 cells were labeled by cultivation in ‘heavy’ medium lacking natural Lys and Arg amino acids supplemented with 13 C 6 lysine and 13 C 6 , 15 N 4 arginine or regular ‘light’ medium ( 12 C-Lys/ 12 C, 14 N-Arg). After density arrest in G 0 , nuclear extracts were prepared. DNA affinity purification was performed with biotinylated cyclin B2 wild-type and CDE/CHR mutant probes. Precipitated proteins were separated by SDS–PAGE, digested with trypsin and analyzed by mass spectrometry.

    Journal: Nucleic Acids Research

    Article Title: The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes

    doi: 10.1093/nar/gkr793

    Figure Lengend Snippet: Strategy for the identification of proteins binding to the CDE/CHR tandem element of the mouse cyclin B2 promoter. NIH3T3 cells were labeled by cultivation in ‘heavy’ medium lacking natural Lys and Arg amino acids supplemented with 13 C 6 lysine and 13 C 6 , 15 N 4 arginine or regular ‘light’ medium ( 12 C-Lys/ 12 C, 14 N-Arg). After density arrest in G 0 , nuclear extracts were prepared. DNA affinity purification was performed with biotinylated cyclin B2 wild-type and CDE/CHR mutant probes. Precipitated proteins were separated by SDS–PAGE, digested with trypsin and analyzed by mass spectrometry.

    Article Snippet: DNA probes for affinity purification with the same sequence as hCCNB2-short and mCcnb2-short and truncated variants of mCcnb2-short without CCAAT-boxes (nt −130 to +1) were obtained by PCR using a biotinylated primer for labeling the 3′-end (Invitrogen, Carlsbad, CA, USA).

    Techniques: Binding Assay, Labeling, Affinity Purification, Mutagenesis, SDS Page, Mass Spectrometry

    eIF4e aptamers inhibit cell proliferation in HeLa and HEK293 cells. HeLa and HEK293 cells were transfected with varying concentrations of aptamers using Lipofectamine 2000 and 2 days after transfection, cell viability was measured with Cell Titer Blue for 3 hours. ( a ) HEK293 cells transfected with 100 nmol/l of each aptamer. NS is an oligonucleotide that can form hairpin structures. ( b ) HEK293 and ( c ) HeLa cells transfected with varying amount of aptamers. ( d ) HEK293 cells transfected with shRNAs against eIF4e or a control shRNA against DMPK TA cloned into pCR2.1. ( e ) HEK293 cells transfected with 100 nmol/l of each aptamer. ( f ) HEK293 cells were transfected with 100 nmol/l of biotinylated Apt 6 or biotinylated NS. Six hours after transfection, cells were fixed with paraformaldehyde and lysed. Cell lysates were then incubated with Streptavidin Dynabeads. Western blots of the input, unbound, and bound fractions were probed with an eIF4e antibody. ** P

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing

    doi: 10.1038/mtna.2014.70

    Figure Lengend Snippet: eIF4e aptamers inhibit cell proliferation in HeLa and HEK293 cells. HeLa and HEK293 cells were transfected with varying concentrations of aptamers using Lipofectamine 2000 and 2 days after transfection, cell viability was measured with Cell Titer Blue for 3 hours. ( a ) HEK293 cells transfected with 100 nmol/l of each aptamer. NS is an oligonucleotide that can form hairpin structures. ( b ) HEK293 and ( c ) HeLa cells transfected with varying amount of aptamers. ( d ) HEK293 cells transfected with shRNAs against eIF4e or a control shRNA against DMPK TA cloned into pCR2.1. ( e ) HEK293 cells transfected with 100 nmol/l of each aptamer. ( f ) HEK293 cells were transfected with 100 nmol/l of biotinylated Apt 6 or biotinylated NS. Six hours after transfection, cells were fixed with paraformaldehyde and lysed. Cell lysates were then incubated with Streptavidin Dynabeads. Western blots of the input, unbound, and bound fractions were probed with an eIF4e antibody. ** P

    Article Snippet: For the pulldown experiments, biotinylated aptamers were purchased from Sigma-Aldrich as oligonucleotides with a 5′-biotin.

    Techniques: Transfection, shRNA, Clone Assay, Incubation, Western Blot

    Selection of DNA aptamers against eIF4e using a rapid selection procedure. ( a ) A library composed of a looped 33-mer random sequence is amplified tenfold by asymmetric PCR with unmodified forward primer and biotinylated reverse primer before negative selection with streptavidin beads and positive selection with eIF4e beads followed by repeating amplification with asymmetric PCR. ( b ) The number of unique sequences identified by high throughput sequencing decreases with increasing rounds of selection. Each round generated between 2 and 3 million valid reads.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing

    doi: 10.1038/mtna.2014.70

    Figure Lengend Snippet: Selection of DNA aptamers against eIF4e using a rapid selection procedure. ( a ) A library composed of a looped 33-mer random sequence is amplified tenfold by asymmetric PCR with unmodified forward primer and biotinylated reverse primer before negative selection with streptavidin beads and positive selection with eIF4e beads followed by repeating amplification with asymmetric PCR. ( b ) The number of unique sequences identified by high throughput sequencing decreases with increasing rounds of selection. Each round generated between 2 and 3 million valid reads.

    Article Snippet: For the pulldown experiments, biotinylated aptamers were purchased from Sigma-Aldrich as oligonucleotides with a 5′-biotin.

    Techniques: Selection, Sequencing, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Generated

    The underlying mechanism of the regulation of miR-138 and LINC00152. ( a ) Amount of LINC00152 bound to SNRNP70 (a positive control), Ago2 or IgG (a negative control) was measured by qPCR after RIP in NOZ cells. ( b ) NOZ cells were transfected with biotinylated NC (Bio-NC), biotinylated wild-type miR-138 (Bio-miR-138) or biotinylated mutant miR-138 (Bio-miR-138-mut), and biotin-based miRNA pull-down assays were conducted after 48 h of transfection. LINC00152 levels were analysed by qPCR. ( c ) Luciferase reporter activity in NOZ cells was detected after co-transfection with pPG-miR-138 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type LINC00152 (pmiR-GLo-LINC00152-wt) or mutant transcripts (pmiR-GLo-LINC00152-mut). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. ( d ) Luciferase reporter activity in NOZ cells was detected after co-transfection with pPG-miR-138 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type HIF-1α (pmiR-GLo-HIF-1α-wt) or mutant transcripts (pmiR-GLo-HIF-1α-mut). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. The mean ± s.d. of triplicate experiments were plotted. ** p

    Journal: Open Biology

    Article Title: Long non-coding RNA LINC00152 promotes gallbladder cancer metastasis and epithelial–mesenchymal transition by regulating HIF-1α via miR-138

    doi: 10.1098/rsob.160247

    Figure Lengend Snippet: The underlying mechanism of the regulation of miR-138 and LINC00152. ( a ) Amount of LINC00152 bound to SNRNP70 (a positive control), Ago2 or IgG (a negative control) was measured by qPCR after RIP in NOZ cells. ( b ) NOZ cells were transfected with biotinylated NC (Bio-NC), biotinylated wild-type miR-138 (Bio-miR-138) or biotinylated mutant miR-138 (Bio-miR-138-mut), and biotin-based miRNA pull-down assays were conducted after 48 h of transfection. LINC00152 levels were analysed by qPCR. ( c ) Luciferase reporter activity in NOZ cells was detected after co-transfection with pPG-miR-138 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type LINC00152 (pmiR-GLo-LINC00152-wt) or mutant transcripts (pmiR-GLo-LINC00152-mut). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. ( d ) Luciferase reporter activity in NOZ cells was detected after co-transfection with pPG-miR-138 (or the empty vector as a control) and the luciferase empty vector (pmiR-GLo), or the vector containing the wild-type HIF-1α (pmiR-GLo-HIF-1α-wt) or mutant transcripts (pmiR-GLo-HIF-1α-mut). Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. The mean ± s.d. of triplicate experiments were plotted. ** p

    Article Snippet: Moreover, we performed miRNA pull-down assays by transfecting biotinylated miR-138, biotinylated miR-138-mut or biotinylated NC into NOZ cells.

    Techniques: Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

    Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).

    Journal: BMC Cancer

    Article Title: Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

    doi: 10.1186/1471-2407-13-476

    Figure Lengend Snippet: Chemotherapy treatment increases ABCC2 expression in ovarian cancer cells. A . qRT-PCR for ABCC2 expression. Ovarian cancer cells treated with LD 50 CBP for 72 hr. OVCAR-5 cells also treated in presence or absence of HA oligomers (HYA oligo, 250 μg/ml). Relative gene expression was determined by calibration against no CBP control and normalized to the house keeping gene β-actin using the 2 -∆∆CT method. Data represents mean ± SEM from 3 independent experiments performed in triplicate. B . OVCAR-5 treated with LD 50 CBP for 72 hr. Fixed cells were incubated with biotinylated HABP and ABCC2 mouse monoclonal antibody and visualized with Cy3-conjugated streptavidin (red) and FITC-conjugated donkey anti-mouse immunoglobulins (green) respectively. Nuclei are counterstained with DAPI dye (blue).

    Article Snippet: OVCAR-5 cells were fixed with cold 100% methanol (5 min) and cold 100% acetone (3 min), washed with PBS and blocked with 5% donkey serum and incubated overnight with biotinylated HABP (2 μg/ml, Seikagaku Corp, Japan) and/or mouse ABCC2 monoclonal antibody (1/25, Clone M2I-4, Abcam, Cambridge, United Kingdom).

    Techniques: Expressing, Quantitative RT-PCR, Incubation

    Validation of TUG1 as a direct target of miR-138-5p (A) The sequences of the predicted miR-138-5p binding site and the TUG1 segments containing the wild type binding site are shown. (B) Dual-luciferase reporter assay revealed that miR-138-5p mimics decreased luciferase activity of pmirGLO-TUG1-Wt, but not of pmirGLO-TUG1-mut. (C) Detection of miR-138-5p using qRT-PCR in the sample pulled down by biotinylated TUG1 probe. (D) miR-138-5p expression in 30 paired of cervical cancer tissues was measured by qRT-PCR. (E) The correlation analysis was performed between TUG1 expression and miR-138-5p in cervical cancer tissues. *P

    Journal: Oncotarget

    Article Title: Long non-coding RNA TUG1 promotes cervical cancer progression by regulating the miR-138-5p-SIRT1 axis

    doi: 10.18632/oncotarget.18224

    Figure Lengend Snippet: Validation of TUG1 as a direct target of miR-138-5p (A) The sequences of the predicted miR-138-5p binding site and the TUG1 segments containing the wild type binding site are shown. (B) Dual-luciferase reporter assay revealed that miR-138-5p mimics decreased luciferase activity of pmirGLO-TUG1-Wt, but not of pmirGLO-TUG1-mut. (C) Detection of miR-138-5p using qRT-PCR in the sample pulled down by biotinylated TUG1 probe. (D) miR-138-5p expression in 30 paired of cervical cancer tissues was measured by qRT-PCR. (E) The correlation analysis was performed between TUG1 expression and miR-138-5p in cervical cancer tissues. *P

    Article Snippet: Pull down assay with biotinylated miRNA Cells were transfected with Biotinylated TUG1 or biotinylated NC (RiboBio) using Lipofectamine 2000.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Expressing

    EGF detection using the immuno-phage assay in 50% BAL fluid VEGF (26 aM - 2.6 pM) was spiked into 50% BAL fluid (in PBS), and incubated with polyclonal anti-VEGF antibody functionalized magnetic particles. The biotinylated antibody/Neutravidin/phage affinity reagent was added, followed by washing. The sample was then analyzed using real-time PCR with phage-specific primers (n=6, error bars = ±1 SD, Non-template control C t ≥34). The No-VEGF control (Ct =22.67±0.55) is shown as a solid line with +/− one standard deviation represented by dotted lines.

    Journal: Biotechnology letters

    Article Title: Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

    doi: 10.1007/s10529-014-1555-9

    Figure Lengend Snippet: EGF detection using the immuno-phage assay in 50% BAL fluid VEGF (26 aM - 2.6 pM) was spiked into 50% BAL fluid (in PBS), and incubated with polyclonal anti-VEGF antibody functionalized magnetic particles. The biotinylated antibody/Neutravidin/phage affinity reagent was added, followed by washing. The sample was then analyzed using real-time PCR with phage-specific primers (n=6, error bars = ±1 SD, Non-template control C t ≥34). The No-VEGF control (Ct =22.67±0.55) is shown as a solid line with +/− one standard deviation represented by dotted lines.

    Article Snippet: To prepare the NeutrAvidin/biotinylated phage construct, NeutrAvidin (A-2666, Life Technologies) and biotinylated phage were incubated at a molar ratio of 100:1 in 500 μl PBS, pH 7.7 on a rotary mixer for 40 min at 25 °C.

    Techniques: Incubation, Real-time Polymerase Chain Reaction, Standard Deviation

    Analyte detection using immuno-phage particles Left panel: Paramagnetic capture particles functionalized with analyte-specific antibodies used to concentrate analyte from solution. Right panel: Pre-made antibody/NeutrAvidin/biotinylated phage affinity agents are detected by real-time PCR of phage DNA; not to scale.

    Journal: Biotechnology letters

    Article Title: Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

    doi: 10.1007/s10529-014-1555-9

    Figure Lengend Snippet: Analyte detection using immuno-phage particles Left panel: Paramagnetic capture particles functionalized with analyte-specific antibodies used to concentrate analyte from solution. Right panel: Pre-made antibody/NeutrAvidin/biotinylated phage affinity agents are detected by real-time PCR of phage DNA; not to scale.

    Article Snippet: To prepare the NeutrAvidin/biotinylated phage construct, NeutrAvidin (A-2666, Life Technologies) and biotinylated phage were incubated at a molar ratio of 100:1 in 500 μl PBS, pH 7.7 on a rotary mixer for 40 min at 25 °C.

    Techniques: Real-time Polymerase Chain Reaction

    (A) PCs express BLyS receptor. Immune BM and spleen (SP) was isolated and stained with αCD138, αB220, and biotinylated BLyS. Histograms of BLyS receptor expression are shown for B220 + CD138 − cells (red); B220 − CD138 − cells (green); and B220 − CD138 + cells (blue). Data are representative of three independent experiments. (B) BM PCs express heightened levels of BCMA mRNA but not BAFF-R or TACI. RNA from splenic B cells (B cells), αCD40-induced B cell blasts (αCD40 B), or BM PCs (PC) was isolated via the TRIzol method. Real-time RT-PCR was performed with each sample to evaluate the relative levels of mRNA expression for BCMA, TACI, and BAFF-R. Data are representative of three experiments. (C) BLyS enhances Mcl-1 expression in PCs but not B cells. Purified CD138 + BM PCs or splenic B cells were cultured with the indicated stimulus, as described in Fig. 1 . After 18 h, RNA was isolated, and the relative expression of antiapoptotic genes was determined relative to β-actin.

    Journal: The Journal of Experimental Medicine

    Article Title: BCMA Is Essential for the Survival of Long-lived Bone Marrow Plasma Cells

    doi: 10.1084/jem.20031330

    Figure Lengend Snippet: (A) PCs express BLyS receptor. Immune BM and spleen (SP) was isolated and stained with αCD138, αB220, and biotinylated BLyS. Histograms of BLyS receptor expression are shown for B220 + CD138 − cells (red); B220 − CD138 − cells (green); and B220 − CD138 + cells (blue). Data are representative of three independent experiments. (B) BM PCs express heightened levels of BCMA mRNA but not BAFF-R or TACI. RNA from splenic B cells (B cells), αCD40-induced B cell blasts (αCD40 B), or BM PCs (PC) was isolated via the TRIzol method. Real-time RT-PCR was performed with each sample to evaluate the relative levels of mRNA expression for BCMA, TACI, and BAFF-R. Data are representative of three experiments. (C) BLyS enhances Mcl-1 expression in PCs but not B cells. Purified CD138 + BM PCs or splenic B cells were cultured with the indicated stimulus, as described in Fig. 1 . After 18 h, RNA was isolated, and the relative expression of antiapoptotic genes was determined relative to β-actin.

    Article Snippet: Incubation with biotinylated antibodies was followed by incubation with streptavidin, peridinine chlorophyll protein, or CyChrome or allophycocyanin (BD Biosciences).

    Techniques: Isolation, Staining, Expressing, Quantitative RT-PCR, Purification, Cell Culture