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  • 99
    New England Biolabs streptavidin magnetic beads
    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through <t>streptavidin</t> purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.
    Streptavidin Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs streptavidin
    A catalytic RNA from the A. pernix genome that reacts with a disubstituted epoxide ( a ) PAGE <t>streptavidin</t> gel mobility shift following incubation of random sequence RNA (“random”), the A. pernix species from Round 6 of the selection (“selected”), and the A. pernix fragment corresponding to the reference genome sequence (“genomic”) with epoxide probe 1 (1 mM) for 16 h at room temperature (1 µM RNA). The complete gel is shown in Supplementary Figure 12 . ( b ) Secondary structure model of the minimized A. pernix catalytic RNA. The reactive guanosine (blue) was identified by RNAse T1 digestion and mass spectrometry. ( c ) Sequence logo based on high-throughput DNA sequencing of the RNA species surviving selection of a partially randomized RNA pool derived from the minimized 42-nt A. pernix catalytic RNA.
    Streptavidin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs hydrophilic streptavidin magnetic beads
    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the <t>streptavidin</t> beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value
    Hydrophilic Streptavidin Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Promega streptavidin coated magnetic beads
    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the <t>streptavidin</t> beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value
    Streptavidin Coated Magnetic Beads, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 877 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher streptavidin coated magnetic microbeads
    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the <t>streptavidin</t> beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value
    Streptavidin Coated Magnetic Microbeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nuclease bal 31
    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the <t>streptavidin</t> beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value
    Nuclease Bal 31, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs m mulv reverse transcriptase
    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the <t>streptavidin</t> beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value
    M Mulv Reverse Transcriptase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Journal: Scientific Reports

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding

    doi: 10.1038/s41598-018-33920-y

    Figure Lengend Snippet: Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Article Snippet: Streptavidin magnetic beads (10 µL, New England BioLabs) were used to precipitate biotinylated species on a magnetic tube rack.

    Techniques: Purification, Western Blot, Expressing, Dominant Negative Mutation, Sequencing, Immunoprecipitation, Over Expression, Inhibition

    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Journal: Biological Procedures Online

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    doi: 10.1007/s12575-009-9022-z

    Figure Lengend Snippet: General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Article Snippet: Streptavidin-coated magnetic beads (1.1 mg/ml) were applied again to purify synthesized double-stranded cDNAs with three times washes.

    Techniques:

    Analysis of Rab prenylation rates in vivo and in vitro . (A-B) Analysis of Rab prenylation status after blocking and releasing prenylation in vivo . HeLa cells were treated with compactin for 24h and then incubated with GGOH for different time periods. Cells were lysed and subjected to in vitro prenylation with BGPP and recombinant RabGGTase and REP for 6h. (A) Degree of prenylation for each Rab was determined by mass spectrometry. The decrease in signal from the timepoint 0h to 5h was determined by label-free spectral counting and converted into the degree of prenylation for each Rab 5 hours after GGOH addition. The graph represents the mean of three independent experiments (±SEM). (B) Streptavidin-HRP Western blot detection of unprenylated Rabs in the cellular lysates at different timepoints after GGOH addition to compactin-treated cells. The cellular lysates were prenylated with BGPP and RabGGTase as in Figure 3 (C) In vitro prenylation of lysate from compactin-treated HeLa cells transfected with different GFP-Rabs. In vitro prenylation reaction was stopped after an hour and subjected to Western blot analysis visualized by infrared Odyssey scanning for total GFP-Rab (GFP/red) and for prenylated biotin-labeled GFP-Rab (biotin/green). Representative blots are shown. (D) The graph represents the percentage of prenylated GFP-Rabs after an hour in vitro prenylation reaction normalized to complete (overnight) prenylation. Means of three independent experiments are shown (±SEM).

    Journal: PLoS ONE

    Article Title: Rab GTPase Prenylation Hierarchy and Its Potential Role in Choroideremia Disease

    doi: 10.1371/journal.pone.0081758

    Figure Lengend Snippet: Analysis of Rab prenylation rates in vivo and in vitro . (A-B) Analysis of Rab prenylation status after blocking and releasing prenylation in vivo . HeLa cells were treated with compactin for 24h and then incubated with GGOH for different time periods. Cells were lysed and subjected to in vitro prenylation with BGPP and recombinant RabGGTase and REP for 6h. (A) Degree of prenylation for each Rab was determined by mass spectrometry. The decrease in signal from the timepoint 0h to 5h was determined by label-free spectral counting and converted into the degree of prenylation for each Rab 5 hours after GGOH addition. The graph represents the mean of three independent experiments (±SEM). (B) Streptavidin-HRP Western blot detection of unprenylated Rabs in the cellular lysates at different timepoints after GGOH addition to compactin-treated cells. The cellular lysates were prenylated with BGPP and RabGGTase as in Figure 3 (C) In vitro prenylation of lysate from compactin-treated HeLa cells transfected with different GFP-Rabs. In vitro prenylation reaction was stopped after an hour and subjected to Western blot analysis visualized by infrared Odyssey scanning for total GFP-Rab (GFP/red) and for prenylated biotin-labeled GFP-Rab (biotin/green). Representative blots are shown. (D) The graph represents the percentage of prenylated GFP-Rabs after an hour in vitro prenylation reaction normalized to complete (overnight) prenylation. Means of three independent experiments are shown (±SEM).

    Article Snippet: Then the lysate was incubated with magnetic streptavidin beads (NEB) for 1 h at room temperature, followed by washes with 1% NP-40, 4 M urea, 4 M guanidine hydrochloride and 40 mM NH4 HCO3 three times each for 10 min at 4°C.

    Techniques: In Vivo, In Vitro, Blocking Assay, Incubation, Recombinant, Mass Spectrometry, Western Blot, Transfection, Labeling

    PRMT5 and MEP50 substrate binding. A. Biotinylated histone peptides [H2A, H2B, H3, H4, and H2A.X-F (all residues 1–20), and H2A.X-F (119–138)] and Npm (176–196) bound to streptavidin beads were incubated with 25 nM (tetramer) PRMT5-MEP50 complex or B. 100 nM MEP50. Captured protein was immunoblotted as indicated. “Beads” indicates no peptide. C. Biotinylated histone peptides [H2A, H2B, H3, H4, and H2A and H4 with phosphorylated S1 (S1ph)] and Npm (176–196) bound to streptavidin beads were incubated with 100 nM (monomer) Flag-HsPRMT5. Captured protein was immunoblotted.

    Journal: PLoS ONE

    Article Title: Structure of the Arginine Methyltransferase PRMT5-MEP50 Reveals a Mechanism for Substrate Specificity

    doi: 10.1371/journal.pone.0057008

    Figure Lengend Snippet: PRMT5 and MEP50 substrate binding. A. Biotinylated histone peptides [H2A, H2B, H3, H4, and H2A.X-F (all residues 1–20), and H2A.X-F (119–138)] and Npm (176–196) bound to streptavidin beads were incubated with 25 nM (tetramer) PRMT5-MEP50 complex or B. 100 nM MEP50. Captured protein was immunoblotted as indicated. “Beads” indicates no peptide. C. Biotinylated histone peptides [H2A, H2B, H3, H4, and H2A and H4 with phosphorylated S1 (S1ph)] and Npm (176–196) bound to streptavidin beads were incubated with 100 nM (monomer) Flag-HsPRMT5. Captured protein was immunoblotted.

    Article Snippet: Peptide Pulldown 5 µg biotinylated peptides were incubated with 20 µl magnetic streptavidin-coupled beads (New England Biolabs, 50% slurry) in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2 HPO4 , 1.47 mM KH2 PO4 , 1 mM PMSF, pH 7.4) for 3 hours at room temperature.

    Techniques: Binding Assay, Incubation

    Selection of streptavidin-binding DeNAno. ( A ) Schematic of selection process. ( B ) Staining of streptavidin selection rounds 1–5. Probe-only, library and positive control (biotinylated library) are also shown. ( C ) Staining of four selected streptavidin clones on streptavidin beads and BSA-coated beads. Negative control clone (G10neg) and biotinylated positive control clone (G10bio) also shown.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Selection of streptavidin-binding DeNAno. ( A ) Schematic of selection process. ( B ) Staining of streptavidin selection rounds 1–5. Probe-only, library and positive control (biotinylated library) are also shown. ( C ) Staining of four selected streptavidin clones on streptavidin beads and BSA-coated beads. Negative control clone (G10neg) and biotinylated positive control clone (G10bio) also shown.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Selection, Binding Assay, Staining, Positive Control, Clone Assay, Negative Control

    Competitive titration and competitive release of streptavidin-binding DeNAno with biotin/biotin derivatives or streptavidin. ( A ) Schematic of competitive titration using biotin/biotin derivatives. ( B ) Free biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. ( C ) Schematic of competitive release using biotin/biotin derivatives. ( D ) Biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline). ( E ) Schematic of streptavidin competitive titration. ( F ) Free streptavidin competition titration of SA-D7 and SA-D8 clones and G10bio positive control. Fluorescently-labeled DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Competitive titration and competitive release of streptavidin-binding DeNAno with biotin/biotin derivatives or streptavidin. ( A ) Schematic of competitive titration using biotin/biotin derivatives. ( B ) Free biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. ( C ) Schematic of competitive release using biotin/biotin derivatives. ( D ) Biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline). ( E ) Schematic of streptavidin competitive titration. ( F ) Free streptavidin competition titration of SA-D7 and SA-D8 clones and G10bio positive control. Fluorescently-labeled DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Titration, Binding Assay, Staining, Clone Assay, Positive Control, Labeling, Incubation

    Dissociation of streptavidin-binding DeNAno over time. Streptavidin-coated magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10 ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and percentage release is graphed (DeNAno in supernatant/DeNAno in total * 100%).

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Dissociation of streptavidin-binding DeNAno over time. Streptavidin-coated magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10 ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and percentage release is graphed (DeNAno in supernatant/DeNAno in total * 100%).

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Binding Assay, Magnetic Beads, Staining, Incubation, Polymerase Chain Reaction

    Imaging of DeNAno and staining different size DeNAno. ( A ) Atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine-coated mica. Scale = 400 nm. ( B ) SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. Scale = 100 nm. ( C ) TEM of small SA-D8 DeNAno roughly 58 nm in diameter. Scale = 100 nm. ( D ) Binding of streptavidin DeNAno of different sizes made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 m at 30°C (the standard conditions), or 93.8 pmol dNTPs for 30 m at 30°C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Imaging of DeNAno and staining different size DeNAno. ( A ) Atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine-coated mica. Scale = 400 nm. ( B ) SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. Scale = 100 nm. ( C ) TEM of small SA-D8 DeNAno roughly 58 nm in diameter. Scale = 100 nm. ( D ) Binding of streptavidin DeNAno of different sizes made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 m at 30°C (the standard conditions), or 93.8 pmol dNTPs for 30 m at 30°C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Imaging, Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Negative Staining, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    Purification and binding assays for His‐tagged LacI variants. (A) Purification of His 6 ‐LacI‐51 and His 6 ‐LacI‐59. Products of purification were examined using SDS‐PAGE to confirm purity (dashed boxes indicate samples used for binding assays). Lane 2 shows trypsin digestion products of wild‐type LacI (no His‐tag) for reference. (B) His‐tagged deletion mutants were used in a pull‐down assay with biotinylated O1 and O scram DNA bound to streptavidin‐coated beads with and without IPTG (I). Note that removal of the hinge helix in His 6 ‐LacI‐59 results in complete loss of DNA binding. (C) Electrophoretic mobility shift assays. Varying concentrations of His 6 ‐LacI‐51 and 10 −7 M LacI were each mixed with [ 32 P]‐labeled 40 bp O1 DNA (≤10 −11 M ) and equilibrated, followed by rapid loading onto a polyacrylamide gel. Note the loss of free DNA and detection of bound bands for His 6 ‐LacI‐51 at 10 −5 M protein; the lowest bound band exhibits slightly higher mobility compared to wild‐type LacI, as expected for this smaller tetramer. Note that wild‐type LacI was separated in this experiment from the His 6 ‐LacI‐51 and O1 DNA, and the dark line indicates removal of wells that contained other materials for analysis. (D) Band intensities for free DNA were derived from phosphorimaging of four separate experiments for His 6 ‐LacI‐51 and three separate experiments for wild‐type LacI; the values were normalized to the free O1 DNA band intensity for each experiment. Standard deviations for free DNA in samples with bound species are indicated.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Lactose repressor hinge domain independently binds DNA

    doi: 10.1002/pro.3372

    Figure Lengend Snippet: Purification and binding assays for His‐tagged LacI variants. (A) Purification of His 6 ‐LacI‐51 and His 6 ‐LacI‐59. Products of purification were examined using SDS‐PAGE to confirm purity (dashed boxes indicate samples used for binding assays). Lane 2 shows trypsin digestion products of wild‐type LacI (no His‐tag) for reference. (B) His‐tagged deletion mutants were used in a pull‐down assay with biotinylated O1 and O scram DNA bound to streptavidin‐coated beads with and without IPTG (I). Note that removal of the hinge helix in His 6 ‐LacI‐59 results in complete loss of DNA binding. (C) Electrophoretic mobility shift assays. Varying concentrations of His 6 ‐LacI‐51 and 10 −7 M LacI were each mixed with [ 32 P]‐labeled 40 bp O1 DNA (≤10 −11 M ) and equilibrated, followed by rapid loading onto a polyacrylamide gel. Note the loss of free DNA and detection of bound bands for His 6 ‐LacI‐51 at 10 −5 M protein; the lowest bound band exhibits slightly higher mobility compared to wild‐type LacI, as expected for this smaller tetramer. Note that wild‐type LacI was separated in this experiment from the His 6 ‐LacI‐51 and O1 DNA, and the dark line indicates removal of wells that contained other materials for analysis. (D) Band intensities for free DNA were derived from phosphorimaging of four separate experiments for His 6 ‐LacI‐51 and three separate experiments for wild‐type LacI; the values were normalized to the free O1 DNA band intensity for each experiment. Standard deviations for free DNA in samples with bound species are indicated.

    Article Snippet: Magnetic bead DNA pull‐down assay Streptavidin magnetic beads (New England BioLabs) were placed in a magnetic tube rack and the supernatant removed.

    Techniques: Purification, Binding Assay, SDS Page, Pull Down Assay, Electrophoretic Mobility Shift Assay, Labeling, Derivative Assay

    A catalytic RNA from the A. pernix genome that reacts with a disubstituted epoxide ( a ) PAGE streptavidin gel mobility shift following incubation of random sequence RNA (“random”), the A. pernix species from Round 6 of the selection (“selected”), and the A. pernix fragment corresponding to the reference genome sequence (“genomic”) with epoxide probe 1 (1 mM) for 16 h at room temperature (1 µM RNA). The complete gel is shown in Supplementary Figure 12 . ( b ) Secondary structure model of the minimized A. pernix catalytic RNA. The reactive guanosine (blue) was identified by RNAse T1 digestion and mass spectrometry. ( c ) Sequence logo based on high-throughput DNA sequencing of the RNA species surviving selection of a partially randomized RNA pool derived from the minimized 42-nt A. pernix catalytic RNA.

    Journal: Nature chemical biology

    Article Title: Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling

    doi: 10.1038/nchembio.1655

    Figure Lengend Snippet: A catalytic RNA from the A. pernix genome that reacts with a disubstituted epoxide ( a ) PAGE streptavidin gel mobility shift following incubation of random sequence RNA (“random”), the A. pernix species from Round 6 of the selection (“selected”), and the A. pernix fragment corresponding to the reference genome sequence (“genomic”) with epoxide probe 1 (1 mM) for 16 h at room temperature (1 µM RNA). The complete gel is shown in Supplementary Figure 12 . ( b ) Secondary structure model of the minimized A. pernix catalytic RNA. The reactive guanosine (blue) was identified by RNAse T1 digestion and mass spectrometry. ( c ) Sequence logo based on high-throughput DNA sequencing of the RNA species surviving selection of a partially randomized RNA pool derived from the minimized 42-nt A. pernix catalytic RNA.

    Article Snippet: The RNA (7 µL) and streptavidin (NEB; 1 µg) were incubated at room temperature for 25 minutes and then combined with gel electrophoresis loading buffer.

    Techniques: Polyacrylamide Gel Electrophoresis, Mobility Shift, Incubation, Sequencing, Selection, Mass Spectrometry, High Throughput Screening Assay, DNA Sequencing, Derivative Assay

    Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins ( a ) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. ( b ) Total RNA from HEK 293T cells was reacted with epoxide-azide 14 , followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14 . ( c ) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1 -modified ASH1 -catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15 .

    Journal: Nature chemical biology

    Article Title: Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling

    doi: 10.1038/nchembio.1655

    Figure Lengend Snippet: Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins ( a ) Transcriptional fusion of a self-labeling catalytic RNA to an RNA of interest may enable selective, covalent RNA modification in a complex biological sample. ( b ) Total RNA from HEK 293T cells was reacted with epoxide-azide 14 , followed by DBCO-TAMRA. Total RNA was analyzed by PAGE and TAMRA-modified RNAs were visualized by fluorescence imaging. Lanes 1 and 6: in vitro transcribed catalytic RNA-fused 5S rRNA containing one or three copies of the catalytic RNA, respectively, rather than cellular RNA. Lanes 2 and 3: the inactive C9-G35 mutant RNA. Lanes 4–8: 5S rRNA fused to one copy (lanes 4–5) or three copies (lanes 6–8) of the active optimized catalytic RNA. Bands at the top of the gel result from incomplete removal of excess DBCO-TAMRA probe or background labeling of cellular rRNAs/mRNAs. The complete gel is shown in Supplementary Figure 14 . ( c ) Western blot probing the presence of three known ASH1 mRNA-binding proteins (Puf6, Khd1, and She2) and one non-binding protein control (Guk1) in yeast cell lysate. Lanes 1 and 2: Lysate incubated overnight with streptavidin-coated magnetic beads only (lane 1) or pre-incubated with 5 µg of epoxide 1 -modified ASH1 -catalytic RNA (lane 2). Unbound proteins were washed away and captured proteins were eluted at 95 °C. Lane 3: Input lysate prior to incubation with beads. The complete gel is shown in Supplementary Figure 15 .

    Article Snippet: The RNA (7 µL) and streptavidin (NEB; 1 µg) were incubated at room temperature for 25 minutes and then combined with gel electrophoresis loading buffer.

    Techniques: RNA Binding Assay, Labeling, Modification, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, In Vitro, Mutagenesis, Western Blot, Binding Assay, Incubation, Magnetic Beads

    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the streptavidin beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value

    Journal: Nature Communications

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria

    doi: 10.1038/s41467-018-05997-6

    Figure Lengend Snippet: SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the streptavidin beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value

    Article Snippet: The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Techniques: RNA Sequencing Assay, Labeling, Expressing

    Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Journal: Nucleic Acids Research

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    doi: 10.1093/nar/gku637

    Figure Lengend Snippet: Overview of 3-day generation of cDNA libraries. ( A ) On the first day, total RNA is ligated to a 3′ adapter and cDNA is generated by reverse transcription by tandem reactions in a single tube, RNA is degraded and cDNAs are isolated by ethanol precipitation. ( B ) On the second day, cDNAs are circularized, size selected by gel fractionation and eluted overnight in the presence of streptavidin beads. ( C ) PCR is done on bead-bound purified cDNAs to generate templates ready for high-throughput sequencing.

    Article Snippet: 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Techniques: Generated, Isolation, Ethanol Precipitation, Fractionation, Polymerase Chain Reaction, Purification, Next-Generation Sequencing

    Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Journal: Nucleic Acids Research

    Article Title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

    doi: 10.1093/nar/gku637

    Figure Lengend Snippet: Detailed LQ cloning method. ( A ) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (N X ), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. ( B ) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides ( C ). Ethanol precipitated cDNAs are circularized ( D ) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads ( E ). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) ( F ). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) ( G ).

    Article Snippet: 5.0 μl magnetic hydrophilic streptavidin beads (New England Biolabs) were washed three times with 50 μl buffer WB (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1.0 mM EDTA), resuspended in 5.0 μl TE + 0.3M NaCl and added to each sample.

    Techniques: Clone Assay, Ligation, Isolation, Polymerase Chain Reaction, Amplification, Generated