Structured Review

Promega streptavidin coated magnetic beads
hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on <t>streptavidin-coated</t> magnetic
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Images

1) Product Images from "Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *"

Article Title: Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.390849

hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic
Figure Legend Snippet: hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic

Techniques Used: Modification

2) Product Images from "FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex"

Article Title: FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1523178113

FtsK stoppage on XerCD/ dif complexes. ( A ) Schematic of the translocation assay using a color code as in . The dotted line represents FtsK translocation, which releases the DNA from the bead (biotine/streptavidin link breakage), allowing its recovery
Figure Legend Snippet: FtsK stoppage on XerCD/ dif complexes. ( A ) Schematic of the translocation assay using a color code as in . The dotted line represents FtsK translocation, which releases the DNA from the bead (biotine/streptavidin link breakage), allowing its recovery

Techniques Used: Translocation Assay

3) Product Images from "DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro"

Article Title: DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2013.137

Aptamers had high binding affinities with U87Δ cells specifically. (A) Binding curves of aptamers 43, 41, 47, and 32. The K d values of all four aptamers were less than 100 nmol/L. This result proved that the selected aptamers had high binding affinities for U87Δ cells. (B) Identification of binding affinity between biotin-aptamer 32 and EGFRvIII protein by Western blot analysis. Lanes (left to right): (1) no aptamer control; (2) biotin-aptamer 32; (3) initial library control. Samples were incubated with streptavidin-magnetic beads respectively, and the complexes were incubated with U87Δ cell lysates. EGFR antibody was used to detect whether the complex could bind EGFRvIII protein.
Figure Legend Snippet: Aptamers had high binding affinities with U87Δ cells specifically. (A) Binding curves of aptamers 43, 41, 47, and 32. The K d values of all four aptamers were less than 100 nmol/L. This result proved that the selected aptamers had high binding affinities for U87Δ cells. (B) Identification of binding affinity between biotin-aptamer 32 and EGFRvIII protein by Western blot analysis. Lanes (left to right): (1) no aptamer control; (2) biotin-aptamer 32; (3) initial library control. Samples were incubated with streptavidin-magnetic beads respectively, and the complexes were incubated with U87Δ cell lysates. EGFR antibody was used to detect whether the complex could bind EGFRvIII protein.

Techniques Used: Binding Assay, Western Blot, Incubation, Magnetic Beads

4) Product Images from "Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase"

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0145872

Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.
Figure Legend Snippet: Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.

Techniques Used: Cell Culture, Incubation, Magnetic Beads, Western Blot, Recombinant, Positive Control

5) Product Images from "Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase"

Article Title: Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase

Journal: Genome Biology

doi: 10.1186/gb-2012-13-8-r69

Comparison between the 5fC-antibody immunoprecipitation and chemical pulldown method . (a) For the 5fC DNA immunoprecipitation we used 1 pg of 5fC-103mer and 10 pg C-103mer in the presence of 5 µg salmon sperm DNA. The immunoprecipitation resulted in an enrichment of 1.6-fold of the 5fC-103mer over the C-103mer. Error bars represent the standard error of the mean. (b) Conditions for the biotinylation reaction of a 9-mer containing a single 5fC. The oligonucleotide was incubated at room temperature with an ARP in the presence of anisidine at pH 5 for 24 h and resulted in the formation of a single product. (c) Pulldown of 1 pg 5fC-biotin-103mer in the presence of 10 pg C-103mer and 5 µg salmon sperm DNA using streptavidin-coated magnetic beads resulted in an enrichment of the biotinylated DNA of around 1,000-fold. Error bars represent the standard error of the mean.
Figure Legend Snippet: Comparison between the 5fC-antibody immunoprecipitation and chemical pulldown method . (a) For the 5fC DNA immunoprecipitation we used 1 pg of 5fC-103mer and 10 pg C-103mer in the presence of 5 µg salmon sperm DNA. The immunoprecipitation resulted in an enrichment of 1.6-fold of the 5fC-103mer over the C-103mer. Error bars represent the standard error of the mean. (b) Conditions for the biotinylation reaction of a 9-mer containing a single 5fC. The oligonucleotide was incubated at room temperature with an ARP in the presence of anisidine at pH 5 for 24 h and resulted in the formation of a single product. (c) Pulldown of 1 pg 5fC-biotin-103mer in the presence of 10 pg C-103mer and 5 µg salmon sperm DNA using streptavidin-coated magnetic beads resulted in an enrichment of the biotinylated DNA of around 1,000-fold. Error bars represent the standard error of the mean.

Techniques Used: Immunoprecipitation, Incubation, Magnetic Beads

6) Product Images from "Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins"

Article Title: Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins

Journal: eLife

doi: 10.7554/eLife.31098

Generation and validation of antibodies to oncogenic KRAS upregulated surface proteins. ( a ) (Left) Schematic of the Fc-fusion construct developed for rapid expression of membrane protein extracellular domains. Each extracellular domain was expressed as a TEV cleavable site-specifically biotinylated Fc-fusion. (Right) HEK293T cells stably expressing an ER-localized biotin ligase are transiently transfected with the Fc-fusion expression vector. Proteins are quantitatively biotinylated in-vivo, secreted into the cellular media, and purified by Protein A affinity purification. ( b ) Shown is the strategy for phage display generation of antibodies to each RAS-induced protein ECD. Proteins were immobilized on streptavidin magnetic beads and mixed with a highly diverse phage-displayed Fab library. Non-binding phage were removed by washing and phage bound protein was released by enzymatic treatment with TEV protease. Eluted phage were propagated in E. coli and the selection process was iterated for 3–4 rounds to enrich the library for specific protein binders. ( c ) Representative phage ELISAs from selections against seven proteins seen elevated in expression level by oncogenic KRAS signaling in MCF10As. Phage clones show strong binding to cognate protein Fc-fusions but little detectable binding to the isolated Fc-domain suggesting binding to the targeted ECD. ( d ) Table of the number of unique antibody clones generated against each of the specified KRAS upregulated targets. ( e ) Representative flow cytometry histograms demonstrate specific cellular target engagement of Fab clones generated against seven KRAS-driven surface proteins. MCF10A cells stably expressing dCas9-KRAB and a decoy sgRNA (red) or target sgRNA (blue and green) were labeled with either a negative control Fab (green) or a Fab of interest (red and blue). Fab binding to cells was detected by addition of a Protein A Alexa647 conjugate and quantification by immunofluorescence flow cytometry.
Figure Legend Snippet: Generation and validation of antibodies to oncogenic KRAS upregulated surface proteins. ( a ) (Left) Schematic of the Fc-fusion construct developed for rapid expression of membrane protein extracellular domains. Each extracellular domain was expressed as a TEV cleavable site-specifically biotinylated Fc-fusion. (Right) HEK293T cells stably expressing an ER-localized biotin ligase are transiently transfected with the Fc-fusion expression vector. Proteins are quantitatively biotinylated in-vivo, secreted into the cellular media, and purified by Protein A affinity purification. ( b ) Shown is the strategy for phage display generation of antibodies to each RAS-induced protein ECD. Proteins were immobilized on streptavidin magnetic beads and mixed with a highly diverse phage-displayed Fab library. Non-binding phage were removed by washing and phage bound protein was released by enzymatic treatment with TEV protease. Eluted phage were propagated in E. coli and the selection process was iterated for 3–4 rounds to enrich the library for specific protein binders. ( c ) Representative phage ELISAs from selections against seven proteins seen elevated in expression level by oncogenic KRAS signaling in MCF10As. Phage clones show strong binding to cognate protein Fc-fusions but little detectable binding to the isolated Fc-domain suggesting binding to the targeted ECD. ( d ) Table of the number of unique antibody clones generated against each of the specified KRAS upregulated targets. ( e ) Representative flow cytometry histograms demonstrate specific cellular target engagement of Fab clones generated against seven KRAS-driven surface proteins. MCF10A cells stably expressing dCas9-KRAB and a decoy sgRNA (red) or target sgRNA (blue and green) were labeled with either a negative control Fab (green) or a Fab of interest (red and blue). Fab binding to cells was detected by addition of a Protein A Alexa647 conjugate and quantification by immunofluorescence flow cytometry.

Techniques Used: Construct, Expressing, Stable Transfection, Transfection, Plasmid Preparation, In Vivo, Purification, Affinity Purification, Magnetic Beads, Binding Assay, Selection, Clone Assay, Isolation, Generated, Flow Cytometry, Cytometry, Labeling, Negative Control, Immunofluorescence

7) Product Images from "Environmental regulation operating at the promoter clearance step of bacterial transcription"

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription

Journal: Genes & Development

doi: 10.1101/gad.1520507

Arg (or CAN) chase of ArgP–RNAP– argO complex assembled in the presence of Lys and NTPs, either in solution ( A ) or on streptavidin-coated beads ( B ). See text for details. Unless otherwise indicated, ArgP was added to all reaction mixes. Other additions were as indicated above the lanes: The primary additives were those that were present in the initial reactions, and the secondary additives were those that were added during the chase step. (A) Arg; (L) Lys; (C) CAN; (RO) run-off transcript from argO promoter; (EE) end-to-end transcription product. In B , the fractions from the primary reaction mixes (pellet [P]; supernatant [S]; unseparated primary reaction mix [P + S]) that were used for preparation of samples for loading on the corresponding lanes of the gel are indicated. Transcription products were resolved on denaturing 6% ( A ) or 10% ( B ) polyacrylamide gels.
Figure Legend Snippet: Arg (or CAN) chase of ArgP–RNAP– argO complex assembled in the presence of Lys and NTPs, either in solution ( A ) or on streptavidin-coated beads ( B ). See text for details. Unless otherwise indicated, ArgP was added to all reaction mixes. Other additions were as indicated above the lanes: The primary additives were those that were present in the initial reactions, and the secondary additives were those that were added during the chase step. (A) Arg; (L) Lys; (C) CAN; (RO) run-off transcript from argO promoter; (EE) end-to-end transcription product. In B , the fractions from the primary reaction mixes (pellet [P]; supernatant [S]; unseparated primary reaction mix [P + S]) that were used for preparation of samples for loading on the corresponding lanes of the gel are indicated. Transcription products were resolved on denaturing 6% ( A ) or 10% ( B ) polyacrylamide gels.

Techniques Used:

8) Product Images from "The Crohn’s disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells"

Article Title: The Crohn’s disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0212850

TCF7L2 binds DNA harboring disease-associated rs6651252 variant with stronger affinity. (A). DNA sequence flanking rs6651252 (asterisk) and the adjacent TCF consensus motif (underlined). (B). Western blot analysis of TCF7L2 from HCT116 protein lysates that were incubated with the biotinylated DNA probes indicated and subsequently precipitated with streptavidin conjugated magnetic beads. A probe containing mutations in the consensus TCF binding element (mutant TBE) was used as a negative control. (C). as in (B) except the biotinylated probe harboring the ancestral T variant was used in all reactions. As indicated below, increasing concentrations of unlabeled probes containing T or C were included in the binding reactions prior to precipitation.
Figure Legend Snippet: TCF7L2 binds DNA harboring disease-associated rs6651252 variant with stronger affinity. (A). DNA sequence flanking rs6651252 (asterisk) and the adjacent TCF consensus motif (underlined). (B). Western blot analysis of TCF7L2 from HCT116 protein lysates that were incubated with the biotinylated DNA probes indicated and subsequently precipitated with streptavidin conjugated magnetic beads. A probe containing mutations in the consensus TCF binding element (mutant TBE) was used as a negative control. (C). as in (B) except the biotinylated probe harboring the ancestral T variant was used in all reactions. As indicated below, increasing concentrations of unlabeled probes containing T or C were included in the binding reactions prior to precipitation.

Techniques Used: Variant Assay, Sequencing, Western Blot, Incubation, Magnetic Beads, Binding Assay, Mutagenesis, Negative Control

9) Product Images from "Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *"

Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.730135

NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′
Figure Legend Snippet: NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′

Techniques Used:

10) Product Images from "Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli"

Article Title: Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163057

riRNA promoter-directed transcription in vitro . [A] Multiple-round transcription in vitro was performed using template I ( Table 1 , probe T1) in the absence and presence of 50 nM Rho (lanes 1 and 2). In the absence of termination factor Rho, three run-off products were indicated, of which the size was estimated to be about 2,400 nt (template-sized run-off transcript), 1,300 nt and 800 nt, respectively. In the presence of Rho, termination was detected at various positions besides the three Rho-independent terminations. Radio-labelled DNA markers are shown to calculate the transcripts size (marker lanes). [B] Transcription in vitro was performed using T7A1 promoter template II ( Table 1 , probe T2). Transcription was performed in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of 50 nM Rho. The DNA template was 5’ biotinylated and was immobilized on Streptavidin-coated magnetic beads to assess the released transcripts. S and P denote the supernatant and pellet fractions, respectively. Discrete-sized transcripts were detected in the supernatant fraction including released transcripts.
Figure Legend Snippet: riRNA promoter-directed transcription in vitro . [A] Multiple-round transcription in vitro was performed using template I ( Table 1 , probe T1) in the absence and presence of 50 nM Rho (lanes 1 and 2). In the absence of termination factor Rho, three run-off products were indicated, of which the size was estimated to be about 2,400 nt (template-sized run-off transcript), 1,300 nt and 800 nt, respectively. In the presence of Rho, termination was detected at various positions besides the three Rho-independent terminations. Radio-labelled DNA markers are shown to calculate the transcripts size (marker lanes). [B] Transcription in vitro was performed using T7A1 promoter template II ( Table 1 , probe T2). Transcription was performed in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of 50 nM Rho. The DNA template was 5’ biotinylated and was immobilized on Streptavidin-coated magnetic beads to assess the released transcripts. S and P denote the supernatant and pellet fractions, respectively. Discrete-sized transcripts were detected in the supernatant fraction including released transcripts.

Techniques Used: In Vitro, Marker, Magnetic Beads

11) Product Images from "Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase"

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0145872

Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.
Figure Legend Snippet: Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.

Techniques Used: Cell Culture, Incubation, Magnetic Beads, Western Blot, Recombinant, Positive Control

12) Product Images from "Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins"

Article Title: Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins

Journal: eLife

doi: 10.7554/eLife.31098

Generation and validation of antibodies to oncogenic KRAS upregulated surface proteins. ( a ) Western blot analysis of Fc-fusion protein endogenous biotinylation. Expression in WT HEK293T cells was compared to expression in HEK293T cells stably expressing BirA localized to the cytosol (Left), the endoplasmic reticulum (Middle), or secreted into the extracellular space (Right). The amount of biotinylation was estimated by assessment of band migration by SDS-PAGE after co-incubation of the purified Fc-fusion with streptavidin. Expression in cells expressing ER-localized BirA showed quantitative biotinylation ( > 98%). ( b ) Phage ELISAs from selections against seven proteins elevated in expression level by oncogenic KRAS signaling in MCF10As. Phage clones that showed strong binding to cognate protein Fc-fusions but little detectable binding to the isolated Fc-domain were advanced for further characterization. ( c ) Schematic of the construct used to display each protein on the surface of HEK293 (T-Rex-293) cells for validation of antibody specificity. ( d ) Representative flow cytometry histograms demonstrate specific cellular target engagement of Fab clones raised against seven RAS-driven surface proteins.
Figure Legend Snippet: Generation and validation of antibodies to oncogenic KRAS upregulated surface proteins. ( a ) Western blot analysis of Fc-fusion protein endogenous biotinylation. Expression in WT HEK293T cells was compared to expression in HEK293T cells stably expressing BirA localized to the cytosol (Left), the endoplasmic reticulum (Middle), or secreted into the extracellular space (Right). The amount of biotinylation was estimated by assessment of band migration by SDS-PAGE after co-incubation of the purified Fc-fusion with streptavidin. Expression in cells expressing ER-localized BirA showed quantitative biotinylation ( > 98%). ( b ) Phage ELISAs from selections against seven proteins elevated in expression level by oncogenic KRAS signaling in MCF10As. Phage clones that showed strong binding to cognate protein Fc-fusions but little detectable binding to the isolated Fc-domain were advanced for further characterization. ( c ) Schematic of the construct used to display each protein on the surface of HEK293 (T-Rex-293) cells for validation of antibody specificity. ( d ) Representative flow cytometry histograms demonstrate specific cellular target engagement of Fab clones raised against seven RAS-driven surface proteins.

Techniques Used: Western Blot, Expressing, Stable Transfection, Migration, SDS Page, Incubation, Purification, Clone Assay, Binding Assay, Isolation, Construct, Flow Cytometry, Cytometry

13) Product Images from "Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase"

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0145872

Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.
Figure Legend Snippet: Pull-down of endogenous Lyn from human cells. Ramos cells were cultured and the clarified lysate was incubated with biotinylated 2H7 or wild-type FN3 (WT-FN3) monobody. Protein complex was then captured with streptavidin-coated magnetic beads and analyzed by Western blot. The Western blot included a recombinant Lyn protein as a positive control, along with the input and output samples of the pull-down experiment. The Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.

Techniques Used: Cell Culture, Incubation, Magnetic Beads, Western Blot, Recombinant, Positive Control

14) Product Images from "Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *"

Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.730135

NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′
Figure Legend Snippet: NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′

Techniques Used:

15) Product Images from "A ribozyme that triphosphorylates RNA 5?-hydroxyl groups"

Article Title: A ribozyme that triphosphorylates RNA 5?-hydroxyl groups

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1405

Scheme for the in vitro selection of triphosphorylation ribozymes. ( A ) A double-stranded DNA library containing the promoter for T7 RNA polymerase (T7), the sequence for a hammerhead ribozyme (HhRz) and a randomized sequence (N150) was transcribed into RNA. ( B ) The 5′-terminal hammerhead ribozyme cleaved itself off cotranscriptionally (filled triangle), generating a 5′-terminal hydroxyl group on the RNA library. ( C ) The RNA library was incubated in the presence of TMP such that active ribozymes could triphosphorylate their 5′-terminus. ( D ) Triphosphorylated RNA molecules were reacted with the 3′-hydroxyl group of a short biotinylated RNA, by the R3C ligase ribozyme. ( E ) Ligated RNAs were captured via their biotin modification on streptavidin-coated magnetic beads and washed stringently. The RNAs were then ( F ) reverse transcribed and ( G ) amplified by PCR to generate the DNA pool for the next round of selection.
Figure Legend Snippet: Scheme for the in vitro selection of triphosphorylation ribozymes. ( A ) A double-stranded DNA library containing the promoter for T7 RNA polymerase (T7), the sequence for a hammerhead ribozyme (HhRz) and a randomized sequence (N150) was transcribed into RNA. ( B ) The 5′-terminal hammerhead ribozyme cleaved itself off cotranscriptionally (filled triangle), generating a 5′-terminal hydroxyl group on the RNA library. ( C ) The RNA library was incubated in the presence of TMP such that active ribozymes could triphosphorylate their 5′-terminus. ( D ) Triphosphorylated RNA molecules were reacted with the 3′-hydroxyl group of a short biotinylated RNA, by the R3C ligase ribozyme. ( E ) Ligated RNAs were captured via their biotin modification on streptavidin-coated magnetic beads and washed stringently. The RNAs were then ( F ) reverse transcribed and ( G ) amplified by PCR to generate the DNA pool for the next round of selection.

Techniques Used: In Vitro, Selection, Sequencing, Incubation, Modification, Magnetic Beads, Amplification, Polymerase Chain Reaction

16) Product Images from "Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease"

Article Title: Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr453

BcgI reactions on isolated DNA. The reactions contained 1 nM BIO-61 ( 32 P-labelled: Figure 1 ) attached to streptavidin-coated beads and 20 nM BcgI REase in Buffer R at 37°C: either no other duplex was present or the solution also contained 5 nM HEX-42S. At various times after adding the enzyme, aliquots were withdrawn, quenched and subjected to denaturing PAGE. The gels were analysed to evaluate the concentrations of the radiolabelled forms of DNA: the intact 61-nt oligonucleotide (the bottom strand of BIO-61) remaining during the reaction in the absence (black squares) and in the presence (white squares) of HEX-42S; and, from the reaction in the presence of HEX-42S, the 14-nt (white triangles) and the 48-nt products (white inverted triangles) from DNA cleavage at the loci proximal to or distal from the 32 P-label, respectively. Error bars denote deviations from the mean of duplicates.
Figure Legend Snippet: BcgI reactions on isolated DNA. The reactions contained 1 nM BIO-61 ( 32 P-labelled: Figure 1 ) attached to streptavidin-coated beads and 20 nM BcgI REase in Buffer R at 37°C: either no other duplex was present or the solution also contained 5 nM HEX-42S. At various times after adding the enzyme, aliquots were withdrawn, quenched and subjected to denaturing PAGE. The gels were analysed to evaluate the concentrations of the radiolabelled forms of DNA: the intact 61-nt oligonucleotide (the bottom strand of BIO-61) remaining during the reaction in the absence (black squares) and in the presence (white squares) of HEX-42S; and, from the reaction in the presence of HEX-42S, the 14-nt (white triangles) and the 48-nt products (white inverted triangles) from DNA cleavage at the loci proximal to or distal from the 32 P-label, respectively. Error bars denote deviations from the mean of duplicates.

Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis

17) Product Images from "Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *"

Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.730135

NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′
Figure Legend Snippet: NPH I translocates 5′ to 3′ on single-stranded DNA. A , phosphorimage of a 10% native polyacrylamide gel of a streptavidin displacement assay. Assays were conducted on 36-mer oligonucleotides that were biotinylated at either the 3′

Techniques Used:

18) Product Images from "ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation"

Article Title: ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation

Journal: eLife

doi: 10.7554/eLife.10150

Structural features of the Prdm14-linker–Mtgr1/Mb (S4) complex. ( A ) Crystals of the Prdm14-linker–Mtgr1/Mb(S4) complex obtained in 16% PEG3350, 8% Tascimate pH 5.3. ( B ) The Prdm14-monobody interface. Prdm14 is shown in white surface representation with the interface residues on Prdm14 shown in yellow and are labeled in black. Mb(S4) is shown in cartoon representation in cyan and are labeled in red. The upper box illustrates prominent features in the interface involving residues in the FG loop of the monobody. Trp81 of Mb(S4) is in the pocket formed by Pro250, Phe270, Val268, Asp262 and Ala265 of Prdm14. Gln75, Tyr77 and Trp81 of Mb(S4) form hydrogen bonds with Prdm14 Arg269, Asp262 and Glu251, respectively. Hydrogen bonds and salt bridges are shown in black dotted lines. The bottom box shows a salt bridge formed between Prdm14 Glu340 and Lys47 in the β-strand C of Mb(S4). ( C ) Unbiased feature-enhanced map showing electron density at the 1 sigma level for residues involved in Prdm14–Mtgr1 interaction. ( D ) Crystal contacts between monobodies and Prdm14 from neighboring molecules. The symmetry related molecules are shown with Prdm14-linker–Mtgr1 in yellow and monobody in cyan. ( E ) Binding of Mb(S4) and its point mutant, Trp81 to Ala, to biotinylated Prdm14 immobilized on streptavidin M280 beads, as measured using the bead-based binding assay. Mutation of Trp81 results in substantial loss of Prdm14 binding, confirming the interface between Prdm14 and Mb(S4) shown in panel B. Mtgr1, myeloid translocation gene related 1. DOI: http://dx.doi.org/10.7554/eLife.10150.022
Figure Legend Snippet: Structural features of the Prdm14-linker–Mtgr1/Mb (S4) complex. ( A ) Crystals of the Prdm14-linker–Mtgr1/Mb(S4) complex obtained in 16% PEG3350, 8% Tascimate pH 5.3. ( B ) The Prdm14-monobody interface. Prdm14 is shown in white surface representation with the interface residues on Prdm14 shown in yellow and are labeled in black. Mb(S4) is shown in cartoon representation in cyan and are labeled in red. The upper box illustrates prominent features in the interface involving residues in the FG loop of the monobody. Trp81 of Mb(S4) is in the pocket formed by Pro250, Phe270, Val268, Asp262 and Ala265 of Prdm14. Gln75, Tyr77 and Trp81 of Mb(S4) form hydrogen bonds with Prdm14 Arg269, Asp262 and Glu251, respectively. Hydrogen bonds and salt bridges are shown in black dotted lines. The bottom box shows a salt bridge formed between Prdm14 Glu340 and Lys47 in the β-strand C of Mb(S4). ( C ) Unbiased feature-enhanced map showing electron density at the 1 sigma level for residues involved in Prdm14–Mtgr1 interaction. ( D ) Crystal contacts between monobodies and Prdm14 from neighboring molecules. The symmetry related molecules are shown with Prdm14-linker–Mtgr1 in yellow and monobody in cyan. ( E ) Binding of Mb(S4) and its point mutant, Trp81 to Ala, to biotinylated Prdm14 immobilized on streptavidin M280 beads, as measured using the bead-based binding assay. Mutation of Trp81 results in substantial loss of Prdm14 binding, confirming the interface between Prdm14 and Mb(S4) shown in panel B. Mtgr1, myeloid translocation gene related 1. DOI: http://dx.doi.org/10.7554/eLife.10150.022

Techniques Used: Labeling, Binding Assay, Mutagenesis, Translocation Assay

19) Product Images from "Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator"

Article Title: Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq450

Antitermination mediated by tiny abortive transcripts. ( A ) Decreasing termination efficiency with increasing reaction times and increasing concentrations of T7 RNA polymerase. In multi-round transcription reactions (20 µl), biotinylated DNA template (50 nM) was pre-incubated with 20, 40 or 80 U of T7 RNA polymerase and 200 µM NTPs at room temperature for varied time periods before radiolabeling of transcripts by incubation with 2 µCi of [α- 32 P]UTP (800 Ci/mmol) for 3 min. Terminated (T) and read-through runoff (R) transcripts were quantified in duplicate, and average termination efficiencies (TE) shown for each lane. ( B ) Plot of experimental data of (A) and abolishment of antitermination due to the prior presence of external C + U-rich RNA. Termination efficiencies ( y -axis) measured in (A) are plotted against reaction time ( x -axis). An experiment (inverted triangles) was additionally performed with 1 µM RNA of ACCCCUU and 80 U of T7 RNA polymerase. ( C ) Diverse transcripts produced at various NTP compositions. All four NTPs were present in the ‘PR1’ experiment, GTP and ATP in ‘PR2’ and GTP only in ‘PR4’, with each NTP at a concentration of 200 µM (++). In ‘PR3’, the concentration of GTP was 200 µM and ATP was 20 µM (+). Biotinylated KM01 template (50 nM) was transcribed with [γ- 32 P]GTP and 80 U of T7 RNA polymerase at room temperature for 30 min. Released RNA transcripts, including short products of various sequences and long terminated (T) and read-through (R) products, were purified using streptavidin-coated magnetic beads and resolved on denaturing 20% polyacrylamide gels. ( D ) Antitermination effects of various RNAs on Tϕ. Termination efficiencies were measured in single-round transcription reactions of radiolabeled ECs stalled at position T−33 with 200 µM NTPs at room temperature in the absence (NC) or presence of nonradioactive transcripts produced in PR1, PR2, PR3 or PR4 experiments. These experiments were additionally performed in the presence of chemically synthesized RNA GGGGG (SR1, a product of reiterative transcription), GGGAGA (SR2, a product of template-dependant transcription) and ACCCCUU (SR3, a sequence in the left bottom of the terminator hairpin). In the last lane, SR3 RNA and PR1 transcripts were together added to the assay.
Figure Legend Snippet: Antitermination mediated by tiny abortive transcripts. ( A ) Decreasing termination efficiency with increasing reaction times and increasing concentrations of T7 RNA polymerase. In multi-round transcription reactions (20 µl), biotinylated DNA template (50 nM) was pre-incubated with 20, 40 or 80 U of T7 RNA polymerase and 200 µM NTPs at room temperature for varied time periods before radiolabeling of transcripts by incubation with 2 µCi of [α- 32 P]UTP (800 Ci/mmol) for 3 min. Terminated (T) and read-through runoff (R) transcripts were quantified in duplicate, and average termination efficiencies (TE) shown for each lane. ( B ) Plot of experimental data of (A) and abolishment of antitermination due to the prior presence of external C + U-rich RNA. Termination efficiencies ( y -axis) measured in (A) are plotted against reaction time ( x -axis). An experiment (inverted triangles) was additionally performed with 1 µM RNA of ACCCCUU and 80 U of T7 RNA polymerase. ( C ) Diverse transcripts produced at various NTP compositions. All four NTPs were present in the ‘PR1’ experiment, GTP and ATP in ‘PR2’ and GTP only in ‘PR4’, with each NTP at a concentration of 200 µM (++). In ‘PR3’, the concentration of GTP was 200 µM and ATP was 20 µM (+). Biotinylated KM01 template (50 nM) was transcribed with [γ- 32 P]GTP and 80 U of T7 RNA polymerase at room temperature for 30 min. Released RNA transcripts, including short products of various sequences and long terminated (T) and read-through (R) products, were purified using streptavidin-coated magnetic beads and resolved on denaturing 20% polyacrylamide gels. ( D ) Antitermination effects of various RNAs on Tϕ. Termination efficiencies were measured in single-round transcription reactions of radiolabeled ECs stalled at position T−33 with 200 µM NTPs at room temperature in the absence (NC) or presence of nonradioactive transcripts produced in PR1, PR2, PR3 or PR4 experiments. These experiments were additionally performed in the presence of chemically synthesized RNA GGGGG (SR1, a product of reiterative transcription), GGGAGA (SR2, a product of template-dependant transcription) and ACCCCUU (SR3, a sequence in the left bottom of the terminator hairpin). In the last lane, SR3 RNA and PR1 transcripts were together added to the assay.

Techniques Used: Incubation, Radioactivity, Produced, Concentration Assay, Purification, Magnetic Beads, Synthesized, Sequencing

20) Product Images from "Single-Molecule Enzymology Based On The Principle Of The Millikan Oil Drop Experiment"

Article Title: Single-Molecule Enzymology Based On The Principle Of The Millikan Oil Drop Experiment

Journal: Analytical biochemistry

doi: 10.1016/j.ab.2013.11.016

System Schematic. a) The tri-functional capture oligonucleotide includes a single stranded oligonucleotide sequence, a disulfide group and a primary amine. B) A schematic of a tethered gold nanoparticle where two 80kDa PEG moieties are linked by streptavidin
Figure Legend Snippet: System Schematic. a) The tri-functional capture oligonucleotide includes a single stranded oligonucleotide sequence, a disulfide group and a primary amine. B) A schematic of a tethered gold nanoparticle where two 80kDa PEG moieties are linked by streptavidin

Techniques Used: Functional Assay, Sequencing

21) Product Images from "A ribozyme that triphosphorylates RNA 5?-hydroxyl groups"

Article Title: A ribozyme that triphosphorylates RNA 5?-hydroxyl groups

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1405

Scheme for the in vitro selection of triphosphorylation ribozymes. ( A ) A double-stranded DNA library containing the promoter for T7 RNA polymerase (T7), the sequence for a hammerhead ribozyme (HhRz) and a randomized sequence (N150) was transcribed into RNA. ( B ) The 5′-terminal hammerhead ribozyme cleaved itself off cotranscriptionally (filled triangle), generating a 5′-terminal hydroxyl group on the RNA library. ( C ) The RNA library was incubated in the presence of TMP such that active ribozymes could triphosphorylate their 5′-terminus. ( D ) Triphosphorylated RNA molecules were reacted with the 3′-hydroxyl group of a short biotinylated RNA, by the R3C ligase ribozyme. ( E ) Ligated RNAs were captured via their biotin modification on streptavidin-coated magnetic beads and washed stringently. The RNAs were then ( F ) reverse transcribed and ( G ) amplified by PCR to generate the DNA pool for the next round of selection.
Figure Legend Snippet: Scheme for the in vitro selection of triphosphorylation ribozymes. ( A ) A double-stranded DNA library containing the promoter for T7 RNA polymerase (T7), the sequence for a hammerhead ribozyme (HhRz) and a randomized sequence (N150) was transcribed into RNA. ( B ) The 5′-terminal hammerhead ribozyme cleaved itself off cotranscriptionally (filled triangle), generating a 5′-terminal hydroxyl group on the RNA library. ( C ) The RNA library was incubated in the presence of TMP such that active ribozymes could triphosphorylate their 5′-terminus. ( D ) Triphosphorylated RNA molecules were reacted with the 3′-hydroxyl group of a short biotinylated RNA, by the R3C ligase ribozyme. ( E ) Ligated RNAs were captured via their biotin modification on streptavidin-coated magnetic beads and washed stringently. The RNAs were then ( F ) reverse transcribed and ( G ) amplified by PCR to generate the DNA pool for the next round of selection.

Techniques Used: In Vitro, Selection, Sequencing, Incubation, Modification, Magnetic Beads, Amplification, Polymerase Chain Reaction

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Adsorption:

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Electrophoresis:

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Article Snippet: The next morning, streptavidin-coated magnetic beads (Promega) were washed three times with PBS, blocked for 30 min with casein (Thermo Fisher Scientific), and added to the mixtures of the biotinylated monobodies and cell lysate. .. The boiled samples, separated from the magnetic beads, and 170 femtomole of recombinant Lyn kinase (Invitrogen; Carlsbad, California, # P2907) were loaded to SDS-PAGE gel for electrophoresis.

Incubation:

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Article Snippet: The final supernatant was divided into two equal aliquots (i.e., 100 μL) and each was mixed with 1.5 nanomole of biotinylated form of either the 2H7 or the wild-type FN3 (WT-FN3) monobody and incubated overnight at 4°C. .. The next morning, streptavidin-coated magnetic beads (Promega) were washed three times with PBS, blocked for 30 min with casein (Thermo Fisher Scientific), and added to the mixtures of the biotinylated monobodies and cell lysate.

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Article Title: Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *
Article Snippet: 1 μg of biotinylated nucleosomes (reconstituted with DNA ligated via an EcoRI site to a biotinylated oligonucleotide) were preincubated with 50 μg of streptavidin-coated magnetic beads (Promega) in binding buffer (10 m m triethanolamine-HCl, 150 m m NaCl, 0.1% v/v Triton X-100, 5% v/v glycerol, 0.1 m m EDTA, pH 7.5) for 4 h at 4 °C. .. Immobilized nucleosomes were incubated with recombinant HP1 protein for 1 h at 4 °C in binding buffer.

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: .. After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads. .. Where indicated, the beads were then separated from the supernatant, washed with transcription buffer containing 0.1 mM Arg (or CAN, as appropriate), and then incubated in the chase step with freshly labeled substrate–heparin mix supplemented with 0.1 mM Arg or CAN for 15 min. Each of the various preparations (i.e., the primary reaction mix before separation of beads from supernatant, the separated supernatant, and the mix obtained following chase reaction on the pellet) was analyzed for labeled transcription products by denaturing gel electrophoresis as described above.

Article Title: FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex
Article Snippet: These DNA molecules were mixed with streptavidin-coated magnetic beads (Streptavidin MagneSphere, Promega), at a ratio of 15 ng of DNA for 1 mL of the commercial beads solution, in 25 mM Tris⋅HCl pH 7.5, 10 mM MgCl2 buffer. .. After 30-min incubation at room temperature, beads were precipitated and washed to eliminate most unbound DNA.

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Article Snippet: .. Streptavidin-coated magnetic beads (Promega, Madison, WI, USA) were incubated with the 5′-biotinylated aptamers for 1 h. The negative control was incubated without aptamers. ..

Article Title: Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase
Article Snippet: .. Following adaptor ligation, DNA and 2 μg poly-dI:dC were incubated with 50 μg streptavidin coated magnetic beads (MagneSphere Promega, Fitchburg, Wisconsin, USA) in 50 μl 2× binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% TWEEN) for 15 minutes at room temperature. .. Beads were washed with 5× 500 μl binding buffer and transferred into a new eppendorf.

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Article Snippet: .. The annealed probes (15 μM) were incubated with 0.1 mg of streptavidin coated magnetic beads (Promega Z5481) for 1.5 h at room temperature. .. Prior to use, the beads were washed three times in 1X sodium citrate buffer (30 mM NaCl, 0.035 mM C6 H5 O7 Na3 ) and then resuspended in 100 μl binding buffer (1M NaCl, 10 mM Tris, 1 mM EDTA, pH = 8.0).

Modification:

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: For the Arg-chase experiments on immobilized template, the argO template fragment from −293 to +109 was prepared by PCR as described above, with the modification that the forward argO primer used was biotinylated at its 5′ end. .. After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads.

Article Title: The Crohn’s disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells
Article Snippet: DNA pull-down assay The DNA pull-down assay was conducted as previously described with minor modification [ ]. .. The annealed probes (15 μM) were incubated with 0.1 mg of streptavidin coated magnetic beads (Promega Z5481) for 1.5 h at room temperature.

Western Blot:

Article Title: DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro
Article Snippet: Streptavidin-coated magnetic beads (Promega, Madison, WI, USA) were incubated with the 5′-biotinylated aptamers for 1 h. The negative control was incubated without aptamers. .. After washing, the resulting samples were dissolved in approximately 50 μL of 2×SDS loading buffer and were evaluated by Western blot analysis.

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase
Article Snippet: .. Protein complex was then pulled down with streptavidin-coated magnetic beads and analyzed by Western blot. .. A recombinant full-length Lyn was included as a positive Western-blot control and the Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.

Translocation Assay:

Article Title: FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex
Article Snippet: Paragraph title: Translocation Test. ... These DNA molecules were mixed with streptavidin-coated magnetic beads (Streptavidin MagneSphere, Promega), at a ratio of 15 ng of DNA for 1 mL of the commercial beads solution, in 25 mM Tris⋅HCl pH 7.5, 10 mM MgCl2 buffer.

Ligation:

Article Title: Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase
Article Snippet: .. Following adaptor ligation, DNA and 2 μg poly-dI:dC were incubated with 50 μg streptavidin coated magnetic beads (MagneSphere Promega, Fitchburg, Wisconsin, USA) in 50 μl 2× binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% TWEEN) for 15 minutes at room temperature. .. Beads were washed with 5× 500 μl binding buffer and transferred into a new eppendorf.

Cell Culture:

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase
Article Snippet: Paragraph title: Cell culturing and pull-down experiment ... The next morning, streptavidin-coated magnetic beads (Promega) were washed three times with PBS, blocked for 30 min with casein (Thermo Fisher Scientific), and added to the mixtures of the biotinylated monobodies and cell lysate.

Sequencing:

Article Title: Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase
Article Snippet: For the chemical pulldown, the ends of the DNA fragments were repaired and paired-end sequencing specific adaptors (Illumina, San Diego, California, U.SA) were ligated using the NEBNext DNA Sample Prep Reagent Set 1 (NEB). .. Following adaptor ligation, DNA and 2 μg poly-dI:dC were incubated with 50 μg streptavidin coated magnetic beads (MagneSphere Promega, Fitchburg, Wisconsin, USA) in 50 μl 2× binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% TWEEN) for 15 minutes at room temperature.

Article Title: Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli
Article Snippet: Samples were mixed with 0.01 ml of formaldehyde loading buffer and loaded onto a 6% sequencing gel and analyzed using FLA 9000 Phosphoimager (GE Healthcare). .. The 5’-biotinylated DNA templates were immobilized onto Streptavidin-coated magnetic beads (Promega).

Sonication:

Article Title: The Crohn’s disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells
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Binding Assay:

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Article Snippet: The annealed probes (15 μM) were incubated with 0.1 mg of streptavidin coated magnetic beads (Promega Z5481) for 1.5 h at room temperature. .. Prior to use, the beads were washed three times in 1X sodium citrate buffer (30 mM NaCl, 0.035 mM C6 H5 O7 Na3 ) and then resuspended in 100 μl binding buffer (1M NaCl, 10 mM Tris, 1 mM EDTA, pH = 8.0).

Nucleic Acid Electrophoresis:

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads. .. Where indicated, the beads were then separated from the supernatant, washed with transcription buffer containing 0.1 mM Arg (or CAN, as appropriate), and then incubated in the chase step with freshly labeled substrate–heparin mix supplemented with 0.1 mM Arg or CAN for 15 min. Each of the various preparations (i.e., the primary reaction mix before separation of beads from supernatant, the separated supernatant, and the mix obtained following chase reaction on the pellet) was analyzed for labeled transcription products by denaturing gel electrophoresis as described above.

Pull Down Assay:

Article Title: The Crohn’s disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells
Article Snippet: Paragraph title: DNA pull-down assay ... The annealed probes (15 μM) were incubated with 0.1 mg of streptavidin coated magnetic beads (Promega Z5481) for 1.5 h at room temperature.

Magnetic Beads:

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Article Title: Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *
Article Snippet: .. 1 μg of biotinylated nucleosomes (reconstituted with DNA ligated via an EcoRI site to a biotinylated oligonucleotide) were preincubated with 50 μg of streptavidin-coated magnetic beads (Promega) in binding buffer (10 m m triethanolamine-HCl, 150 m m NaCl, 0.1% v/v Triton X-100, 5% v/v glycerol, 0.1 m m EDTA, pH 7.5) for 4 h at 4 °C. .. Unbound peptides or nucleosomes were removed by two washes with binding buffer.

Article Title: Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes *
Article Snippet: .. Each dsDNA transcription template was bound to streptavidin-coated magnetic beads through a biotin at the 5′ end of the NT strand. .. The sequence of these oligo-based transcription templates was based on a previously characterized template for vaccinia early transcription called Ter29 ( , , , ).

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: .. After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads. .. Where indicated, the beads were then separated from the supernatant, washed with transcription buffer containing 0.1 mM Arg (or CAN, as appropriate), and then incubated in the chase step with freshly labeled substrate–heparin mix supplemented with 0.1 mM Arg or CAN for 15 min. Each of the various preparations (i.e., the primary reaction mix before separation of beads from supernatant, the separated supernatant, and the mix obtained following chase reaction on the pellet) was analyzed for labeled transcription products by denaturing gel electrophoresis as described above.

Article Title: DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro
Article Snippet: .. Streptavidin-coated magnetic beads (Promega, Madison, WI, USA) were incubated with the 5′-biotinylated aptamers for 1 h. The negative control was incubated without aptamers. ..

Article Title: Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase
Article Snippet: .. Following adaptor ligation, DNA and 2 μg poly-dI:dC were incubated with 50 μg streptavidin coated magnetic beads (MagneSphere Promega, Fitchburg, Wisconsin, USA) in 50 μl 2× binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% TWEEN) for 15 minutes at room temperature. .. Beads were washed with 5× 500 μl binding buffer and transferred into a new eppendorf.

Article Title: The Crohn’s disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells
Article Snippet: .. The annealed probes (15 μM) were incubated with 0.1 mg of streptavidin coated magnetic beads (Promega Z5481) for 1.5 h at room temperature. .. Prior to use, the beads were washed three times in 1X sodium citrate buffer (30 mM NaCl, 0.035 mM C6 H5 O7 Na3 ) and then resuspended in 100 μl binding buffer (1M NaCl, 10 mM Tris, 1 mM EDTA, pH = 8.0).

Article Title: Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli
Article Snippet: .. The 5’-biotinylated DNA templates were immobilized onto Streptavidin-coated magnetic beads (Promega). ..

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase
Article Snippet: .. Protein complex was then pulled down with streptavidin-coated magnetic beads and analyzed by Western blot. .. A recombinant full-length Lyn was included as a positive Western-blot control and the Lyn proteins were detected on the blot with a commercial anti-Lyn antibody.

Isolation:

Article Title: Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli
Article Snippet: The reactions were stopped and RNA products were isolated by phenol extraction followed by ethanol precipitation. .. The 5’-biotinylated DNA templates were immobilized onto Streptavidin-coated magnetic beads (Promega).

Labeling:

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: .. After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads. .. Where indicated, the beads were then separated from the supernatant, washed with transcription buffer containing 0.1 mM Arg (or CAN, as appropriate), and then incubated in the chase step with freshly labeled substrate–heparin mix supplemented with 0.1 mM Arg or CAN for 15 min. Each of the various preparations (i.e., the primary reaction mix before separation of beads from supernatant, the separated supernatant, and the mix obtained following chase reaction on the pellet) was analyzed for labeled transcription products by denaturing gel electrophoresis as described above.

Polymerase Chain Reaction:

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: For the Arg-chase experiments on immobilized template, the argO template fragment from −293 to +109 was prepared by PCR as described above, with the modification that the forward argO primer used was biotinylated at its 5′ end. .. After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads.

SDS Page:

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase
Article Snippet: The next morning, streptavidin-coated magnetic beads (Promega) were washed three times with PBS, blocked for 30 min with casein (Thermo Fisher Scientific), and added to the mixtures of the biotinylated monobodies and cell lysate. .. The boiled samples, separated from the magnetic beads, and 170 femtomole of recombinant Lyn kinase (Invitrogen; Carlsbad, California, # P2907) were loaded to SDS-PAGE gel for electrophoresis.

Negative Control:

Article Title: DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro
Article Snippet: .. Streptavidin-coated magnetic beads (Promega, Madison, WI, USA) were incubated with the 5′-biotinylated aptamers for 1 h. The negative control was incubated without aptamers. ..

Selection:

Article Title: Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins
Article Snippet: Briefly, selections with antibody phage Library F ( ) were performed using biotinylated Fc-fusion antigens captured with streptavidin-coated magnetic beads (Promega). .. Prior to each selection, the phage pool was incubated with 1 µM of biotinylated Fc-domain immobilized on streptavidin beads in order to deplete the library of any binders to the beads or Fc-tag.

Sample Prep:

Article Title: Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase
Article Snippet: For the chemical pulldown, the ends of the DNA fragments were repaired and paired-end sequencing specific adaptors (Illumina, San Diego, California, U.SA) were ligated using the NEBNext DNA Sample Prep Reagent Set 1 (NEB). .. Following adaptor ligation, DNA and 2 μg poly-dI:dC were incubated with 50 μg streptavidin coated magnetic beads (MagneSphere Promega, Fitchburg, Wisconsin, USA) in 50 μl 2× binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.1% TWEEN) for 15 minutes at room temperature.

In Vitro:

Article Title: Environmental regulation operating at the promoter clearance step of bacterial transcription
Article Snippet: Paragraph title: In vitro transcription ... After preincubation of template with RNAP (and other additives as appropriate) as above, labeled substrate–heparin mix was added together with streptavidin-coated magnetic beads (Promega Corp.) and the primary reaction mixtures were incubated for 15 min to allow for immobilization of the biotinylated template on the beads.

Article Title: Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli
Article Snippet: Paragraph title: Transcription in vitro ... The 5’-biotinylated DNA templates were immobilized onto Streptavidin-coated magnetic beads (Promega).

Ethanol Precipitation:

Article Title: Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli
Article Snippet: The reactions were stopped and RNA products were isolated by phenol extraction followed by ethanol precipitation. .. The 5’-biotinylated DNA templates were immobilized onto Streptavidin-coated magnetic beads (Promega).

Acid Assay:

Article Title: DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro
Article Snippet: Pull-down assays using streptavidin-magnetic beads The U87Δ cell lysates were quantified using the bicinchoninic acid assay with 600 μg of total protein per sample. .. Streptavidin-coated magnetic beads (Promega, Madison, WI, USA) were incubated with the 5′-biotinylated aptamers for 1 h. The negative control was incubated without aptamers.

Recombinant:

Article Title: Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase
Article Snippet: The next morning, streptavidin-coated magnetic beads (Promega) were washed three times with PBS, blocked for 30 min with casein (Thermo Fisher Scientific), and added to the mixtures of the biotinylated monobodies and cell lysate. .. The boiled samples, separated from the magnetic beads, and 170 femtomole of recombinant Lyn kinase (Invitrogen; Carlsbad, California, # P2907) were loaded to SDS-PAGE gel for electrophoresis.

Article Title: Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *
Article Snippet: 1 μg of biotinylated nucleosomes (reconstituted with DNA ligated via an EcoRI site to a biotinylated oligonucleotide) were preincubated with 50 μg of streptavidin-coated magnetic beads (Promega) in binding buffer (10 m m triethanolamine-HCl, 150 m m NaCl, 0.1% v/v Triton X-100, 5% v/v glycerol, 0.1 m m EDTA, pH 7.5) for 4 h at 4 °C. .. Immobilized nucleosomes were incubated with recombinant HP1 protein for 1 h at 4 °C in binding buffer.

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    Promega streptavidin coated magnetic beads
    hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on <t>streptavidin-coated</t> magnetic
    Streptavidin Coated Magnetic Beads, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic

    Journal: The Journal of Biological Chemistry

    Article Title: Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *

    doi: 10.1074/jbc.M112.390849

    Figure Lengend Snippet: hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic

    Article Snippet: 1 μg of biotinylated nucleosomes (reconstituted with DNA ligated via an EcoRI site to a biotinylated oligonucleotide) were preincubated with 50 μg of streptavidin-coated magnetic beads (Promega) in binding buffer (10 m m triethanolamine-HCl, 150 m m NaCl, 0.1% v/v Triton X-100, 5% v/v glycerol, 0.1 m m EDTA, pH 7.5) for 4 h at 4 °C.

    Techniques: Modification

    FtsK stoppage on XerCD/ dif complexes. ( A ) Schematic of the translocation assay using a color code as in . The dotted line represents FtsK translocation, which releases the DNA from the bead (biotine/streptavidin link breakage), allowing its recovery

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex

    doi: 10.1073/pnas.1523178113

    Figure Lengend Snippet: FtsK stoppage on XerCD/ dif complexes. ( A ) Schematic of the translocation assay using a color code as in . The dotted line represents FtsK translocation, which releases the DNA from the bead (biotine/streptavidin link breakage), allowing its recovery

    Article Snippet: These DNA molecules were mixed with streptavidin-coated magnetic beads (Streptavidin MagneSphere, Promega), at a ratio of 15 ng of DNA for 1 mL of the commercial beads solution, in 25 mM Tris⋅HCl pH 7.5, 10 mM MgCl2 buffer.

    Techniques: Translocation Assay

    Aptamers had high binding affinities with U87Δ cells specifically. (A) Binding curves of aptamers 43, 41, 47, and 32. The K d values of all four aptamers were less than 100 nmol/L. This result proved that the selected aptamers had high binding affinities for U87Δ cells. (B) Identification of binding affinity between biotin-aptamer 32 and EGFRvIII protein by Western blot analysis. Lanes (left to right): (1) no aptamer control; (2) biotin-aptamer 32; (3) initial library control. Samples were incubated with streptavidin-magnetic beads respectively, and the complexes were incubated with U87Δ cell lysates. EGFR antibody was used to detect whether the complex could bind EGFRvIII protein.

    Journal: Acta Pharmacologica Sinica

    Article Title: DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro

    doi: 10.1038/aps.2013.137

    Figure Lengend Snippet: Aptamers had high binding affinities with U87Δ cells specifically. (A) Binding curves of aptamers 43, 41, 47, and 32. The K d values of all four aptamers were less than 100 nmol/L. This result proved that the selected aptamers had high binding affinities for U87Δ cells. (B) Identification of binding affinity between biotin-aptamer 32 and EGFRvIII protein by Western blot analysis. Lanes (left to right): (1) no aptamer control; (2) biotin-aptamer 32; (3) initial library control. Samples were incubated with streptavidin-magnetic beads respectively, and the complexes were incubated with U87Δ cell lysates. EGFR antibody was used to detect whether the complex could bind EGFRvIII protein.

    Article Snippet: Streptavidin-coated magnetic beads (Promega, Madison, WI, USA) were incubated with the 5′-biotinylated aptamers for 1 h. The negative control was incubated without aptamers.

    Techniques: Binding Assay, Western Blot, Incubation, Magnetic Beads