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  • 99
    New England Biolabs t4 polynucleotide kinase
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare nitrocellulose membrane
    Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a <t>Hybond-N+</t> membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.
    Nitrocellulose Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 45054 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare nylon membrane
    Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a <t>Hybond-N+</t> membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.
    Nylon Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 17892 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare hybond ecl nitrocellulose membrane
    Hsp90 protein binds PS/cEt or PS/LNA ASOs but not PS/MOE ASO. ( A ) Affinity selection was performed using biotinylated gapmer PS-ASOs with different 2′-modifications in the wings. Proteins were eluted using non-biotinylated PS-ASOs with the same chemical composition. Isolated proteins were separated by SDS-PAGE, and visualized by silver staining (upper panel), or detected by Western assay for a duplicate gel (lower panel) using antibodies specific to Hsp90α or HSP90β. Ku70 protein served as a control for loading. The protein band containing Hsp90 is indicated for the silver stained gel. ( B ) Western analysis for Hsp90 protein co-selected using a biotinylated PS/cEt ASO, and eluted by competition using PS/cEt ASOs with two different sequences that target PTEN or NCL1 mRNAs, or using a PS/MOE ASO targeting NCL1. The Hsp90 antibody recognizes both α and β isoforms. Ku70 protein was detected and served as a control. ( C ) Affinity selection using PS/cEt ASOs that have the same sequence and chemistry but tagged with biotin at either 5′ or 3′ end. Co-isolated Hsp90 and Ku70 were detected by western assay. ( D ) Affinity selection was carried out using either a single stranded PS/cEt ASO or a duplex formed using the same ASO and a complementary 2′- O -methylated oligonucleotide. Co-isolated proteins were directly separated on SDS-PAGE and analyzed by Western assay for Hsp90 and Ku70 proteins. ( E ) Affinity selection was conducted using either a PS/MOE ASO 386652 or a PS/cEt ASO 586183. After washing, bound proteins were first eluted by RNase I (5 U/μl) or DNase I (5 U/μl) treatment for 30 min at RT, followed by elution using 9M Urea from the same beads for 30 min at RT. The eluted proteins were precipitated and analyzed by Western assay for Hsp90 protein. ( F ) Silver staining of one μg of the recombinant Hsp90α protein (Abcam, ab80369). ( G ) Interaction of the recombinant Hsp90α protein with PS/cEt ASO was determined by affinity selection using biotinylated PS-ASOs and 3 μg of purified Hsp90 protein. After wash, the beads were directly loaded on a 4–12% SDS-PAGE, and Hsp90 protein was detected by Western assay. The ASO ID numbers in each panel are shown. The above experiments were repeated at least three times and representative results are shown. ( H ) Membrane binding assay for Hsp90α protein and different PS-ASOs, as described in Materials and Methods. For each ASO, triplicate binding reactions were performed. The molar ratio between Hsp90a protein and ASO was given below the wells. An example of a double-filter binding for PS/cEt and PS/LNA ASOs is shown in the middle panel for protein-bound ASOs captured using a <t>Hybond</t> <t>ECL</t> membrane, and the lower panel for unbound ASOs captured using a Hybond-N+ nylon membrane. The signal intensity for the ASOs was quantified and the binding curves for ( I ) PS/cEt and ( J ) PS/LNA ASOs were plotted using Prism. The calculated kds are given. The error bars represent standard deviations from three experiments.
    Hybond Ecl Nitrocellulose Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 4637 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    GE Healthcare pvdf membrane
    Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% <t>SDS–polyacrylamide</t> gel, electroblotted to <t>PVDF</t> filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.
    Pvdf Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 13849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare polyvinylidene fluoride pvdf membrane
    Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% <t>SDS–polyacrylamide</t> gel, electroblotted to <t>PVDF</t> filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.
    Polyvinylidene Fluoride Pvdf Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare ecl
    Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% <t>SDS–polyacrylamide</t> gel, electroblotted to <t>PVDF</t> filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.
    Ecl, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 31041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a Hybond-N+ membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.

    Journal: The Journal of surgical research

    Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION

    doi: 10.1016/j.jss.2009.03.072

    Figure Lengend Snippet: Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a Hybond-N+ membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.

    Article Snippet: For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX).

    Techniques: Autoradiography, Northern Blot, Isolation, Multiple Displacement Amplification, Labeling, Real-time Polymerase Chain Reaction, Negative Control

    Noncoding RNAs connected with the flv4-2 operon. A , schematic presentation of chromosomal arrangement of the genes flv4 , sll 0218, and flv2 in the flv4-2 operon; ctpB ; the antisense transcribed sections for as1_flv4 , as2_flv4 , and as_flv2 ; and the gene for ncRNA, ncr0080. B , verification of transcript accumulation by Northern blot analysis. RNA was isolated from HC-grown Synechocystis WT cells, separated by denaturing electrophoresis, and blotted onto a Hybond-N nylon membrane. RNA was stained with methylene blue. Specific radiolabeled probes were derived from in vitro transcription. Arrows indicate the selected signals. M , molecular mass marker; R , total RNA after electrophoresis. C , classification of the noncoding RNA Ncr0080. Shown is the region in between the start codons for ctpB (168533–168535) and flv4 (168152–168150). The ncr0080 sequence is in gray boldface , and the transcription start for flv4 , according to the results of 5′-rapid amplification of cDNA end experiments, is marked with an arrow . Predicted −10 and −35 elements are underlined . The chromosomal positions are according to Cyanobase.

    Journal: The Journal of Biological Chemistry

    Article Title: The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply *

    doi: 10.1074/jbc.M112.391755

    Figure Lengend Snippet: Noncoding RNAs connected with the flv4-2 operon. A , schematic presentation of chromosomal arrangement of the genes flv4 , sll 0218, and flv2 in the flv4-2 operon; ctpB ; the antisense transcribed sections for as1_flv4 , as2_flv4 , and as_flv2 ; and the gene for ncRNA, ncr0080. B , verification of transcript accumulation by Northern blot analysis. RNA was isolated from HC-grown Synechocystis WT cells, separated by denaturing electrophoresis, and blotted onto a Hybond-N nylon membrane. RNA was stained with methylene blue. Specific radiolabeled probes were derived from in vitro transcription. Arrows indicate the selected signals. M , molecular mass marker; R , total RNA after electrophoresis. C , classification of the noncoding RNA Ncr0080. Shown is the region in between the start codons for ctpB (168533–168535) and flv4 (168152–168150). The ncr0080 sequence is in gray boldface , and the transcription start for flv4 , according to the results of 5′-rapid amplification of cDNA end experiments, is marked with an arrow . Predicted −10 and −35 elements are underlined . The chromosomal positions are according to Cyanobase.

    Article Snippet: To characterize small RNAs, RNA samples (5 μg) were mixed with RNA loading dye (Fermentas, St. Leon-Rot, Germany), denatured for 10 min at 70 °C, separated in 10% polyacrylamide-urea gels, and transferred to Hybond-N nylon membranes (GE Healthcare) by electroblotting for 1 h. For the mRNA studies, RNA samples were mixed with denaturation solution, incubated for 10 min at 70 °C, separated in 1.3% agarose gels containing 7% formaldehyde in MOPS, and transferred to Hybond-N nylon membranes by capillary blotting with 10× SSC overnight ( ).

    Techniques: Northern Blot, Isolation, Electrophoresis, Staining, Derivative Assay, In Vitro, Marker, Sequencing, Amplification

    Expression and localization of NRP/B in neuronal origin-derived cells and human brain specimen. A , for Northern blot analysis, total RNA was purified from HCT-116, SH-SY5Y, and PC12 cells using TRIzol reagent. Ten μg of RNA was blotted on a Hybond-N + membrane and hybridized with an α- 32 P-labeled human NRP/B probe. B , total cell lysates were prepared from several cell lines, and 100-μg samples were blotted on a polyvinylidene difluoride membrane. The blot was incubated with anti-NRP/B (VD2) or anti-c-src tyrosine kinase ( CSK ) antibodies. CSK was used to confirm equal protein loading. C , localization of NRP/B in sample specimens of normal brains. Normal brain specimens were obtained from the Cooperative Human Tissue Network. The paraffin-embedded samples were immunostained with anti-NRP/B (VD2) antibody. WB , Western blot.

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Matrix Protein (NRP/B) Modulates the Nuclear Factor (Erythroid-derived 2)-related 2 (NRF2)-dependent Oxidative Stress Response *

    doi: 10.1074/jbc.M109.095786

    Figure Lengend Snippet: Expression and localization of NRP/B in neuronal origin-derived cells and human brain specimen. A , for Northern blot analysis, total RNA was purified from HCT-116, SH-SY5Y, and PC12 cells using TRIzol reagent. Ten μg of RNA was blotted on a Hybond-N + membrane and hybridized with an α- 32 P-labeled human NRP/B probe. B , total cell lysates were prepared from several cell lines, and 100-μg samples were blotted on a polyvinylidene difluoride membrane. The blot was incubated with anti-NRP/B (VD2) or anti-c-src tyrosine kinase ( CSK ) antibodies. CSK was used to confirm equal protein loading. C , localization of NRP/B in sample specimens of normal brains. Normal brain specimens were obtained from the Cooperative Human Tissue Network. The paraffin-embedded samples were immunostained with anti-NRP/B (VD2) antibody. WB , Western blot.

    Article Snippet: 20 μg of total RNA was electrophoresed on a 1% agarose gel, and transferred onto Hybond-N+ (Amersham Biosciences) in 20× SSC buffer overnight.

    Techniques: Expressing, Derivative Assay, Northern Blot, Purification, Labeling, Incubation, Western Blot

    Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the Hybond-P PVDF membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.

    Journal: Scientific Reports

    Article Title: Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC)

    doi: 10.1038/srep10897

    Figure Lengend Snippet: Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the Hybond-P PVDF membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.

    Article Snippet: Then, 1 μL of each aptamer-LCN2 complex was spotted onto a Hybond-P PVDF membrane (GE Healthcare, Uppsala, Sweden).

    Techniques: Binding Assay, Incubation, Software

    Caspase-3 inhibitor labels several processed forms of recombinant AtCathB3  in vitro  and C 131 A abolishes labelling. ( a ) Cartoon of the recombinant AtCathB3 expressed in insect cells and its various processed forms: cherrytag (tag); prodomain (ProD); catalytic domain (catalytic); C-terminal prodomain (CD); his-tag in blue; the position the catalytic aa are indicated including C 131  (Q,C,H,N). ( b ) Recombinant AtCathB3 wild-type and mutant C 131 A were affinity purified and partially activated at 70  μ g/ml, pH 8.0 or pH 5.3, at 4 °C. Aliquots were separated using SDS-PAGE and visualised by instant blue (top), incubated with anti-his antibody, ECL on western blot (middle) or activity labelling at pH 5 by 25  μ M biotin-DEVD–FMK and detected using streptavidin-HRP on western blot (bottom). ( c ) Recombinant AtCathB3 was partially activated at 200  μ g/ml, pH 5.5, at 8 °C, separated on a 15% SDS-PAGE and visualised using Coomassie blue (Coomassie) or transferred on Hybond P, incubated with anti-his antibody, followed by ECL detection. There were two processed forms: P and ΔC. N-terminal sequencing of P indicated a N-terminal at G27 of the native sequence ( Supplementary Figure S2 ). ( d ) Full activation and de-glycosylation of AtCathB3: a fully processed form (M) was obtained at pH 4.5, 1 mg/ml, 10  μ g/ml dextran. Activated aliquots were labelled using 25  μ M biotin-DEVD–FMK (−). In addition, one labelled aliquot was deglycosylated using PNGase for 1 h at 37 °C (+). Labelled proteins were visualised by ECL detection on western blot using streptavidin-HRP.

    Journal: Cell Death and Differentiation

    Article Title: Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis

    doi: 10.1038/cdd.2016.34

    Figure Lengend Snippet: Caspase-3 inhibitor labels several processed forms of recombinant AtCathB3 in vitro and C 131 A abolishes labelling. ( a ) Cartoon of the recombinant AtCathB3 expressed in insect cells and its various processed forms: cherrytag (tag); prodomain (ProD); catalytic domain (catalytic); C-terminal prodomain (CD); his-tag in blue; the position the catalytic aa are indicated including C 131 (Q,C,H,N). ( b ) Recombinant AtCathB3 wild-type and mutant C 131 A were affinity purified and partially activated at 70  μ g/ml, pH 8.0 or pH 5.3, at 4 °C. Aliquots were separated using SDS-PAGE and visualised by instant blue (top), incubated with anti-his antibody, ECL on western blot (middle) or activity labelling at pH 5 by 25  μ M biotin-DEVD–FMK and detected using streptavidin-HRP on western blot (bottom). ( c ) Recombinant AtCathB3 was partially activated at 200  μ g/ml, pH 5.5, at 8 °C, separated on a 15% SDS-PAGE and visualised using Coomassie blue (Coomassie) or transferred on Hybond P, incubated with anti-his antibody, followed by ECL detection. There were two processed forms: P and ΔC. N-terminal sequencing of P indicated a N-terminal at G27 of the native sequence ( Supplementary Figure S2 ). ( d ) Full activation and de-glycosylation of AtCathB3: a fully processed form (M) was obtained at pH 4.5, 1 mg/ml, 10  μ g/ml dextran. Activated aliquots were labelled using 25  μ M biotin-DEVD–FMK (−). In addition, one labelled aliquot was deglycosylated using PNGase for 1 h at 37 °C (+). Labelled proteins were visualised by ECL detection on western blot using streptavidin-HRP.

    Article Snippet: Western blot and activity labelling Proteins were separated on SDS-PAGE (15%) and transferred to a Hybond-P membrane (Amersham Biosciences) at 100 V for 1 h in transfer buffer.

    Techniques: Recombinant, In Vitro, Mutagenesis, Affinity Purification, SDS Page, Incubation, Western Blot, Activity Assay, Sequencing, Activation Assay

    Detection of smRNAs. Low-molecular weight RNA fractions were blotted on a Hybond-XL membrane and hybridized with the hydrolyzed sense-specific npt II riboprobe. A 20 nt oligomer was used as a size marker. The loading control was a non-specific high molecular band. Approximately 50 µg of RNA material was loaded into each lane. SR1, non-transgenic control; HeLo2, non-silenced line; HoLo1, PTGS single locus line; HeLo1E, TGS single locus line; Lo1/Lo2 and Lo1/LoB, PTGS F1 hybrids; Lo1E/Lo2 and Lo1E/LoB, non-silenced F1 hybrids.

    Journal: Nucleic Acids Research

    Article Title: The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco

    doi: 10.1093/nar/gkl180

    Figure Lengend Snippet: Detection of smRNAs. Low-molecular weight RNA fractions were blotted on a Hybond-XL membrane and hybridized with the hydrolyzed sense-specific npt II riboprobe. A 20 nt oligomer was used as a size marker. The loading control was a non-specific high molecular band. Approximately 50 µg of RNA material was loaded into each lane. SR1, non-transgenic control; HeLo2, non-silenced line; HoLo1, PTGS single locus line; HeLo1E, TGS single locus line; Lo1/Lo2 and Lo1/LoB, PTGS F1 hybrids; Lo1E/Lo2 and Lo1E/LoB, non-silenced F1 hybrids.

    Article Snippet: The RNA was denatured in 0.05 M NaOH and blotted onto a Hybond-XL membrane (GE-Healthcare) in 20× SSC.

    Techniques: Molecular Weight, Marker, Transgenic Assay

    Immunoblot analysis of selected PSRPs . An aliquot of 50 μg soluble proteins was separated by 12.5% SDS–PAGE, blotted onto Hybond-C membrane, processed for one-dimensional immunoblot analysis and quantified by densitometry. Immunoreactive protein levels for DnaK and MSR was detected using the respective primary antibodies and signals were detected. U represents unstressed fruit. Commassie blue-stained gel indicates equal protein loading across time points.

    Journal: Frontiers in Plant Science

    Article Title: Proteometabolomic Study of Compatible Interaction in Tomato Fruit Challenged with Sclerotinia rolfsii Illustrates Novel Protein Network during Disease Progression

    doi: 10.3389/fpls.2016.01034

    Figure Lengend Snippet: Immunoblot analysis of selected PSRPs . An aliquot of 50 μg soluble proteins was separated by 12.5% SDS–PAGE, blotted onto Hybond-C membrane, processed for one-dimensional immunoblot analysis and quantified by densitometry. Immunoreactive protein levels for DnaK and MSR was detected using the respective primary antibodies and signals were detected. U represents unstressed fruit. Commassie blue-stained gel indicates equal protein loading across time points.

    Article Snippet: The electrophoresis was performed at room temperature and the proteins were electroblotted onto Hybond-C membrane (Amersham Biosciences, U.K.) at 150 mA for 2 h. The membranes were blocked with 5% (w/v) nonfat milk in TBST buffer (0.1 M Tris pH 7.9, 0.15 M NaCl and 0.1% Tween 20) and probed with primary polyclonal anti-MSR (ab16803) and anti-DnaK (ab80161) (Abcam Ltd., U.K.) at varying dilutions (1:1000–1:15000) in TBS buffer.

    Techniques: SDS Page, Staining

    Viral protein synthesis in A549 cells. A549 cells were infected with wild-type or mutant viruses at a multiplicity of 200 particles per cell. Cells were infected for 9 h (top) or 18 h (bottom) and lysed in RIPA lysis buffer. Proteins were separated by SDS-12.5% PAGE, transferred to Hybond-P, and probed with the antibodies indicated on the left. The proteins were visualized with ECL reagents.

    Journal: Journal of Virology

    Article Title: Distinct Roles of the Adenovirus E4 ORF3 Protein in Viral DNA Replication and Inhibition of Genome Concatenation

    doi: 10.1128/JVI.77.9.5295-5304.2003

    Figure Lengend Snippet: Viral protein synthesis in A549 cells. A549 cells were infected with wild-type or mutant viruses at a multiplicity of 200 particles per cell. Cells were infected for 9 h (top) or 18 h (bottom) and lysed in RIPA lysis buffer. Proteins were separated by SDS-12.5% PAGE, transferred to Hybond-P, and probed with the antibodies indicated on the left. The proteins were visualized with ECL reagents.

    Article Snippet: Twenty micrograms of total protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a Hybond-P (Amersham) membrane, and probed with the appropriate antibody.

    Techniques: Infection, Mutagenesis, Lysis, Polyacrylamide Gel Electrophoresis

    Hsp90 protein binds PS/cEt or PS/LNA ASOs but not PS/MOE ASO. ( A ) Affinity selection was performed using biotinylated gapmer PS-ASOs with different 2′-modifications in the wings. Proteins were eluted using non-biotinylated PS-ASOs with the same chemical composition. Isolated proteins were separated by SDS-PAGE, and visualized by silver staining (upper panel), or detected by Western assay for a duplicate gel (lower panel) using antibodies specific to Hsp90α or HSP90β. Ku70 protein served as a control for loading. The protein band containing Hsp90 is indicated for the silver stained gel. ( B ) Western analysis for Hsp90 protein co-selected using a biotinylated PS/cEt ASO, and eluted by competition using PS/cEt ASOs with two different sequences that target PTEN or NCL1 mRNAs, or using a PS/MOE ASO targeting NCL1. The Hsp90 antibody recognizes both α and β isoforms. Ku70 protein was detected and served as a control. ( C ) Affinity selection using PS/cEt ASOs that have the same sequence and chemistry but tagged with biotin at either 5′ or 3′ end. Co-isolated Hsp90 and Ku70 were detected by western assay. ( D ) Affinity selection was carried out using either a single stranded PS/cEt ASO or a duplex formed using the same ASO and a complementary 2′- O -methylated oligonucleotide. Co-isolated proteins were directly separated on SDS-PAGE and analyzed by Western assay for Hsp90 and Ku70 proteins. ( E ) Affinity selection was conducted using either a PS/MOE ASO 386652 or a PS/cEt ASO 586183. After washing, bound proteins were first eluted by RNase I (5 U/μl) or DNase I (5 U/μl) treatment for 30 min at RT, followed by elution using 9M Urea from the same beads for 30 min at RT. The eluted proteins were precipitated and analyzed by Western assay for Hsp90 protein. ( F ) Silver staining of one μg of the recombinant Hsp90α protein (Abcam, ab80369). ( G ) Interaction of the recombinant Hsp90α protein with PS/cEt ASO was determined by affinity selection using biotinylated PS-ASOs and 3 μg of purified Hsp90 protein. After wash, the beads were directly loaded on a 4–12% SDS-PAGE, and Hsp90 protein was detected by Western assay. The ASO ID numbers in each panel are shown. The above experiments were repeated at least three times and representative results are shown. ( H ) Membrane binding assay for Hsp90α protein and different PS-ASOs, as described in Materials and Methods. For each ASO, triplicate binding reactions were performed. The molar ratio between Hsp90a protein and ASO was given below the wells. An example of a double-filter binding for PS/cEt and PS/LNA ASOs is shown in the middle panel for protein-bound ASOs captured using a Hybond ECL membrane, and the lower panel for unbound ASOs captured using a Hybond-N+ nylon membrane. The signal intensity for the ASOs was quantified and the binding curves for ( I ) PS/cEt and ( J ) PS/LNA ASOs were plotted using Prism. The calculated kds are given. The error bars represent standard deviations from three experiments.

    Journal: Nucleic Acids Research

    Article Title: Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2′-modifications and enhances antisense activity

    doi: 10.1093/nar/gkw144

    Figure Lengend Snippet: Hsp90 protein binds PS/cEt or PS/LNA ASOs but not PS/MOE ASO. ( A ) Affinity selection was performed using biotinylated gapmer PS-ASOs with different 2′-modifications in the wings. Proteins were eluted using non-biotinylated PS-ASOs with the same chemical composition. Isolated proteins were separated by SDS-PAGE, and visualized by silver staining (upper panel), or detected by Western assay for a duplicate gel (lower panel) using antibodies specific to Hsp90α or HSP90β. Ku70 protein served as a control for loading. The protein band containing Hsp90 is indicated for the silver stained gel. ( B ) Western analysis for Hsp90 protein co-selected using a biotinylated PS/cEt ASO, and eluted by competition using PS/cEt ASOs with two different sequences that target PTEN or NCL1 mRNAs, or using a PS/MOE ASO targeting NCL1. The Hsp90 antibody recognizes both α and β isoforms. Ku70 protein was detected and served as a control. ( C ) Affinity selection using PS/cEt ASOs that have the same sequence and chemistry but tagged with biotin at either 5′ or 3′ end. Co-isolated Hsp90 and Ku70 were detected by western assay. ( D ) Affinity selection was carried out using either a single stranded PS/cEt ASO or a duplex formed using the same ASO and a complementary 2′- O -methylated oligonucleotide. Co-isolated proteins were directly separated on SDS-PAGE and analyzed by Western assay for Hsp90 and Ku70 proteins. ( E ) Affinity selection was conducted using either a PS/MOE ASO 386652 or a PS/cEt ASO 586183. After washing, bound proteins were first eluted by RNase I (5 U/μl) or DNase I (5 U/μl) treatment for 30 min at RT, followed by elution using 9M Urea from the same beads for 30 min at RT. The eluted proteins were precipitated and analyzed by Western assay for Hsp90 protein. ( F ) Silver staining of one μg of the recombinant Hsp90α protein (Abcam, ab80369). ( G ) Interaction of the recombinant Hsp90α protein with PS/cEt ASO was determined by affinity selection using biotinylated PS-ASOs and 3 μg of purified Hsp90 protein. After wash, the beads were directly loaded on a 4–12% SDS-PAGE, and Hsp90 protein was detected by Western assay. The ASO ID numbers in each panel are shown. The above experiments were repeated at least three times and representative results are shown. ( H ) Membrane binding assay for Hsp90α protein and different PS-ASOs, as described in Materials and Methods. For each ASO, triplicate binding reactions were performed. The molar ratio between Hsp90a protein and ASO was given below the wells. An example of a double-filter binding for PS/cEt and PS/LNA ASOs is shown in the middle panel for protein-bound ASOs captured using a Hybond ECL membrane, and the lower panel for unbound ASOs captured using a Hybond-N+ nylon membrane. The signal intensity for the ASOs was quantified and the binding curves for ( I ) PS/cEt and ( J ) PS/LNA ASOs were plotted using Prism. The calculated kds are given. The error bars represent standard deviations from three experiments.

    Article Snippet: After 1 h incubation at 37°C, the samples were loaded on a Hybond ECL nitrocellulose membrane (GE Healthcare) pre-treated for 1 h with wash buffer (20 mM Tris.Cl, pH 7.5; 1 M NaCl) in a 96-well dot-blot apparatus (Bio-Rad Bio-Dot), and the protein-bound ASOs were transferred to the membrane by applying vacuum.

    Techniques: Allele-specific Oligonucleotide, Selection, Isolation, SDS Page, Silver Staining, Western Blot, Staining, Sequencing, Methylation, Recombinant, Purification, Binding Assay

    Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% SDS–polyacrylamide gel, electroblotted to PVDF filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.

    Journal: Nucleic Acids Research

    Article Title: The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription

    doi: 10.1093/nar/gkl181

    Figure Lengend Snippet: Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% SDS–polyacrylamide gel, electroblotted to PVDF filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.

    Article Snippet: Total and mitochondrial lysates, containing 250 µg of proteins, were fractionated on 12% SDS–polyacrylamide gel and electroblotted for 1 h to PVDF membrane (Hybond-P, Amersham Pharmacia).

    Techniques: Western Blot, Incubation, Recombinant