dynabeads m 280 streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher dynabeads m 280 streptavidin
    Mutational analyses of the MCAT motif and MCAT-like sequences by reporter and in vitro DNA pulldown assays. (A) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of the expression plasmid for TEAD4 (pCMV-HA-TEAD4) and the Renilla luciferase plasmid. Two days after transfection, the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. The levels of luciferase activity are presented as fold increase compared with that obtained from cells transfected with pA3B−200/+45 without the TEAD4 expression plasmid. The data are the averages of three independent experiments, with the error bars representing standard deviations. (B) Schematic representation of the biotinylated DNA probes used in DNA pulldown assays. The positions and nucleotide sequences of MCAT-like 1 and 2 and MCAT are shown. The mutated nucleotides are underlined, and the nucleotide sequences in the mutated probes are denoted by lowercase letters. (C to F) The indicated biotinylated DNA probes were coupled to <t>Dynabeads/M-280</t> <t>streptavidin</t> and incubated with the nuclear extract from NIKS-E6; 1/10 or 1/20 volume of input (Input) and entire precipitated fractions were analyzed by immunoblotting using anti-pan-TEAD antibody. (E) Unlabeled oligonucleotide competitors (lines A to D) indicated by the solid lines under the nucleotide sequence of the −141/−96 probe were added to the reaction mixtures, and inhibition of TEADs binding to the −141/−96 probe was examined. The MCAT-like sequences 1 and 2 are boxed, and nucleotides that match the typical MCAT motif are underlined. (G) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of pCMV-HA-TEAD4 and the Renilla luciferase plasmid, and the luciferase activity was measured as described for panel A.
    Dynabeads M 280 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 264 article reviews
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    dynabeads m 280 streptavidin - by Bioz Stars, 2019-12
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    Images

    1) Product Images from "Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor"

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor

    Journal:

    doi: 10.1128/JVI.02413-16

    Mutational analyses of the MCAT motif and MCAT-like sequences by reporter and in vitro DNA pulldown assays. (A) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of the expression plasmid for TEAD4 (pCMV-HA-TEAD4) and the Renilla luciferase plasmid. Two days after transfection, the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. The levels of luciferase activity are presented as fold increase compared with that obtained from cells transfected with pA3B−200/+45 without the TEAD4 expression plasmid. The data are the averages of three independent experiments, with the error bars representing standard deviations. (B) Schematic representation of the biotinylated DNA probes used in DNA pulldown assays. The positions and nucleotide sequences of MCAT-like 1 and 2 and MCAT are shown. The mutated nucleotides are underlined, and the nucleotide sequences in the mutated probes are denoted by lowercase letters. (C to F) The indicated biotinylated DNA probes were coupled to Dynabeads/M-280 streptavidin and incubated with the nuclear extract from NIKS-E6; 1/10 or 1/20 volume of input (Input) and entire precipitated fractions were analyzed by immunoblotting using anti-pan-TEAD antibody. (E) Unlabeled oligonucleotide competitors (lines A to D) indicated by the solid lines under the nucleotide sequence of the −141/−96 probe were added to the reaction mixtures, and inhibition of TEADs binding to the −141/−96 probe was examined. The MCAT-like sequences 1 and 2 are boxed, and nucleotides that match the typical MCAT motif are underlined. (G) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of pCMV-HA-TEAD4 and the Renilla luciferase plasmid, and the luciferase activity was measured as described for panel A.
    Figure Legend Snippet: Mutational analyses of the MCAT motif and MCAT-like sequences by reporter and in vitro DNA pulldown assays. (A) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of the expression plasmid for TEAD4 (pCMV-HA-TEAD4) and the Renilla luciferase plasmid. Two days after transfection, the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. The levels of luciferase activity are presented as fold increase compared with that obtained from cells transfected with pA3B−200/+45 without the TEAD4 expression plasmid. The data are the averages of three independent experiments, with the error bars representing standard deviations. (B) Schematic representation of the biotinylated DNA probes used in DNA pulldown assays. The positions and nucleotide sequences of MCAT-like 1 and 2 and MCAT are shown. The mutated nucleotides are underlined, and the nucleotide sequences in the mutated probes are denoted by lowercase letters. (C to F) The indicated biotinylated DNA probes were coupled to Dynabeads/M-280 streptavidin and incubated with the nuclear extract from NIKS-E6; 1/10 or 1/20 volume of input (Input) and entire precipitated fractions were analyzed by immunoblotting using anti-pan-TEAD antibody. (E) Unlabeled oligonucleotide competitors (lines A to D) indicated by the solid lines under the nucleotide sequence of the −141/−96 probe were added to the reaction mixtures, and inhibition of TEADs binding to the −141/−96 probe was examined. The MCAT-like sequences 1 and 2 are boxed, and nucleotides that match the typical MCAT motif are underlined. (G) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of pCMV-HA-TEAD4 and the Renilla luciferase plasmid, and the luciferase activity was measured as described for panel A.

    Techniques Used: In Vitro, Transfection, Luciferase, Plasmid Preparation, Expressing, Hemagglutination Assay, Activity Assay, Incubation, Sequencing, Inhibition, Binding Assay

    2) Product Images from "Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN"

    Article Title: Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

    Journal:

    doi: 10.5966/sctm.2012-0154

    Podocalyxin is a glycoprotein ligand of rBC2LCN. Dynabeads M-280 streptavidin (10 μl) immobilized with biotinylated anti-podocalyxin pAb (1 μg) was incubated with hydrophilic fractions of cells at 4°C overnight with agitation.
    Figure Legend Snippet: Podocalyxin is a glycoprotein ligand of rBC2LCN. Dynabeads M-280 streptavidin (10 μl) immobilized with biotinylated anti-podocalyxin pAb (1 μg) was incubated with hydrophilic fractions of cells at 4°C overnight with agitation.

    Techniques Used: Incubation

    3) Product Images from "Host Heterogeneous Ribonucleoprotein K (hnRNP K) as a Potential Target to Suppress Hepatitis B Virus ReplicationTargeting of a Host Protein to Suppress Hepatitis B Virus Replication"

    Article Title: Host Heterogeneous Ribonucleoprotein K (hnRNP K) as a Potential Target to Suppress Hepatitis B Virus ReplicationTargeting of a Host Protein to Suppress Hepatitis B Virus Replication

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0020163

    Evidence for the Involvement of a Host Cellular Protein in Enh II Activity and HBV Replication (A) Electrophoretic mobility shift assays were performed using HepG2 nuclear extracts with four different probes. Probe 1, lanes 1–4; probe 2 (1752A), lanes 5–8; probe 3, lanes 9–12; probe 4 (1752G), lanes 13–16. Each set of probes contains increasing concentrations (0.0 μg, 0.05 μg, 0.10 μg, and 0.15 μg) of non-specific competitor DNA [poly-(dI)-poly-(dC)], respectively. (B) 40 μg of nuclear protein extracts obtained from HepG2 cells was allowed to bind onto 5 mg Dynabeads M-280 streptavidin-biotin-oligonucleotides in the presence of 2:1 (w/w) ratio of non-specific competitor DNA poly (dI–dC). 1-D isoelectric focusing was followed by 2-D vertical separation on SDS-PAGE (10%). The estimated molecular weight of the specific protein spots detected by silver staining (arrow) is indicated.
    Figure Legend Snippet: Evidence for the Involvement of a Host Cellular Protein in Enh II Activity and HBV Replication (A) Electrophoretic mobility shift assays were performed using HepG2 nuclear extracts with four different probes. Probe 1, lanes 1–4; probe 2 (1752A), lanes 5–8; probe 3, lanes 9–12; probe 4 (1752G), lanes 13–16. Each set of probes contains increasing concentrations (0.0 μg, 0.05 μg, 0.10 μg, and 0.15 μg) of non-specific competitor DNA [poly-(dI)-poly-(dC)], respectively. (B) 40 μg of nuclear protein extracts obtained from HepG2 cells was allowed to bind onto 5 mg Dynabeads M-280 streptavidin-biotin-oligonucleotides in the presence of 2:1 (w/w) ratio of non-specific competitor DNA poly (dI–dC). 1-D isoelectric focusing was followed by 2-D vertical separation on SDS-PAGE (10%). The estimated molecular weight of the specific protein spots detected by silver staining (arrow) is indicated.

    Techniques Used: Activity Assay, Electrophoretic Mobility Shift Assay, SDS Page, Molecular Weight, Silver Staining

    4) Product Images from "KRIBB11 Inhibits HSP70 Synthesis through Inhibition of Heat Shock Factor 1 Function by Impairing the Recruitment of Positive Transcription Elongation Factor b to the hsp70 Promoter"

    Article Title: KRIBB11 Inhibits HSP70 Synthesis through Inhibition of Heat Shock Factor 1 Function by Impairing the Recruitment of Positive Transcription Elongation Factor b to the hsp70 Promoter

    Journal:

    doi: 10.1074/jbc.M110.179440

    KRIBB11 binds to HSF1. A, structure of biotinyl-KRIBB11. B, 300 μg of total protein was incubated with 10 μ m biotinyl-KRIBB11 and competed with KRIBB11 at an indicated concentration. Proteins were captured with 10 μl of Dynabeads M-280 streptavidin and eluted by boiling in SDS-PAGE sample buffer. The eluted proteins were resolved by 10% SDS-PAGE and blotted with an anti-HSF1-specific antibody.
    Figure Legend Snippet: KRIBB11 binds to HSF1. A, structure of biotinyl-KRIBB11. B, 300 μg of total protein was incubated with 10 μ m biotinyl-KRIBB11 and competed with KRIBB11 at an indicated concentration. Proteins were captured with 10 μl of Dynabeads M-280 streptavidin and eluted by boiling in SDS-PAGE sample buffer. The eluted proteins were resolved by 10% SDS-PAGE and blotted with an anti-HSF1-specific antibody.

    Techniques Used: Incubation, Concentration Assay, SDS Page

    5) Product Images from "The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus"

    Article Title: The Use of NanoTrap Particles as a Sample Enrichment Method to Enhance the Detection of Rift Valley Fever Virus

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002296

    Comparison of capture and RNase degradation protection of commercially available beads with NT53. A) RVFV was incubated with NT53 (1), Dynabeads M-280 Streptavidin (2), Biorex 70 Resin (3), Bio-gel HTP Hydroxyapatite (4), SP Sephadex C-25 (5), Sephacryl S-200 beads (6), or DEAE-Sephadex (7) for 30 minutes at room temperature. The sample was washed 4 times with water and then particles were tested in plaque assays to determine how much virus was bound by the particles. B) NT53 or HTP was incubated with purified RNA at 1.0E+7 genomic copies for 30 minutes at room temperature. Following water washes, the samples were resuspended in water and treated with 380 Units/ml of RNase A. Samples with no RNase A treatment were processed in parallel. The viral RNA was extracted from isolated particles and quantitated by qRT-PCR (black bars). Samples without NT53 were processed in parallel (gray bars). C) NT53 and HTP beads were incubated with viral supernatants containing RVFV at 1.7E+8 pfu/ml for 30 minutes at room temperature. Samples were then inactivated by incubation at 57°C for one hour or incubating in the presence of 1% NP-40 at room temperature for 1 hour. A “no bead” control processed in parallel was included for each condition. Viral RNA was extracted from the particles and quantitated by qRT-PCR.
    Figure Legend Snippet: Comparison of capture and RNase degradation protection of commercially available beads with NT53. A) RVFV was incubated with NT53 (1), Dynabeads M-280 Streptavidin (2), Biorex 70 Resin (3), Bio-gel HTP Hydroxyapatite (4), SP Sephadex C-25 (5), Sephacryl S-200 beads (6), or DEAE-Sephadex (7) for 30 minutes at room temperature. The sample was washed 4 times with water and then particles were tested in plaque assays to determine how much virus was bound by the particles. B) NT53 or HTP was incubated with purified RNA at 1.0E+7 genomic copies for 30 minutes at room temperature. Following water washes, the samples were resuspended in water and treated with 380 Units/ml of RNase A. Samples with no RNase A treatment were processed in parallel. The viral RNA was extracted from isolated particles and quantitated by qRT-PCR (black bars). Samples without NT53 were processed in parallel (gray bars). C) NT53 and HTP beads were incubated with viral supernatants containing RVFV at 1.7E+8 pfu/ml for 30 minutes at room temperature. Samples were then inactivated by incubation at 57°C for one hour or incubating in the presence of 1% NP-40 at room temperature for 1 hour. A “no bead” control processed in parallel was included for each condition. Viral RNA was extracted from the particles and quantitated by qRT-PCR.

    Techniques Used: Incubation, Purification, Isolation, Quantitative RT-PCR

    6) Product Images from "Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection"

    Article Title: Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17050585

    Scheme of DNA nanoconstruct of biotin-modified oligonucleotides bound to E6-HPV16 oncogene joined to streptavidin modified MPs : ( A ) 100 µL (10 mg·mL −1 ) of commercial Dynabeads M-280 Streptavidin MPs; ( B ) E6-HPV16 complementary oligonucleotides biotinylated (forward and reverse) (20 µL, 100 µM) using Biotin 3′ end DNA Labeling Kit (Thermo Scientific, Waltham, MA, USA) were successfully conjugated with Dynabeads; and ( C ) E6-HPV16 DNA was amplified from E6-HPV16-pUC57 synthetic plasmid by PCR, which was subsequently purified and conjugated with the nanoconstruct.
    Figure Legend Snippet: Scheme of DNA nanoconstruct of biotin-modified oligonucleotides bound to E6-HPV16 oncogene joined to streptavidin modified MPs : ( A ) 100 µL (10 mg·mL −1 ) of commercial Dynabeads M-280 Streptavidin MPs; ( B ) E6-HPV16 complementary oligonucleotides biotinylated (forward and reverse) (20 µL, 100 µM) using Biotin 3′ end DNA Labeling Kit (Thermo Scientific, Waltham, MA, USA) were successfully conjugated with Dynabeads; and ( C ) E6-HPV16 DNA was amplified from E6-HPV16-pUC57 synthetic plasmid by PCR, which was subsequently purified and conjugated with the nanoconstruct.

    Techniques Used: Modification, DNA Labeling, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Purification

    Related Articles

    Clone Assay:

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: For construction of the pBSAd20 plasmid, the first two leader exons of the adenovirus major late transcription unit, separated by a shortened version of the first intervening sequence, were cloned into the pBS- vector (Stratagene) under the T7 promoter. .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions.

    Centrifugation:

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen). .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage
    Article Snippet: Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose. .. Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose.

    Amplification:

    Article Title: A tissue-specific landscape of sense/antisense transcription in the mouse intestine
    Article Snippet: 13 cycles of amplification led to a yield of more than 2 µg cDNA. .. Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment.

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: PCR amplification conditions were as follows: 2 min at 95°C; 4 cycles of 15 s at 95 °C, 30 s at 60°C, and 30 s at 72°C; then 5 min at 72°C. .. We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen).

    Synthesized:

    Article Title: Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX
    Article Snippet: Dynabeads M-280 Streptavidin was purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA). .. Dynabeads M-280 Streptavidin was purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: Templates were synthesized by PCR from pBSAd20 or pBSAd22 using biotinylated, upstream primer Ade3 (5′-biotin-GTTGGGTAACGCCAGGG-3′) and downstream primer T3-L (5′-GCGCAATTAACCCTCACTAAA-3′). .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions.

    Blocking Assay:

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: In parallel, the 300 ng of each DNA library were pooled with the blocker #1, #2, and #3 reagents (Agilent), denatured for 5 min at 95°C, and then incubated at 65°C in a thermal cycler (MJ Research). .. We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen). .. The reaction product was then purified with the MinElute PCR purification kit (Qiagen) according to the manufacturer’s protocol.

    SYBR Green Assay:

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Incubation:

    Article Title: An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair
    Article Snippet: After ether extraction to remove the unincorporated AAAF, the DNA was precipitated in ethanol, dried, and resuspended in TE (10 m m Tris acetate, pH 7.5, 1 m m EDTA). .. To immobilize the DNA, 300 μl of Dynabeads M-280 streptavidin (Invitrogen) were prewashed in PBS containing 1 mg/ml BSA and 0.05% Nonidet P-40 and then incubated with 15 μg of biotinylated DNA in 10 m m Tris, 1 m m EDTA, 1 m NaCl. .. Typical yields were ∼40–50 ng of DNA per μl of Dynabeads.

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Article Title: The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage
    Article Snippet: Extracts were supplemented with 2 mM MgCl2 and benzonase and incubated for 30 min at 4°C. .. Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose.

    Article Title: Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins
    Article Snippet: One microgram of paramagnetic polystyrene microspheres covalently linked to streptavidin (Dynabeads M-280 Streptavidin; Dynal, Great Neck, NY) was washed twice in diethyl pyrocarbonate-treated solution A (0.1 M NaOH/0.05 M NaCl) and once in diethyl pyrocarbonate-treated solution B (0.1 M NaOH) by placing the tube in a magnetic particle concentrator for 3 min and then removing the free fraction, to ensure an RNase-free environment. .. For immunopurification of ELAV-like proteins together with the bound mRNAs, 10 μg of biotinylated 16A11 mAb was added to the beads in PBS + 1% BSA and incubated for 30 min at room temperature.

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
    Article Snippet: Pulldown was performed with RNA oligonucleotide [2′- O -methylated phosphorothioate with tetra-ethyleneglycol biotin (TEG-Biotin) on 3′-end], having 20 nucleotides complementary to the first antisense box of SNORD27 RNA. .. M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension. .. Nuclear supernatant corresponding to five 15-cm Petri dishes of the HeLa cells in ST2M+0.1NP40 buffer was depleted from unspecific streptavidin binding material by incubation with prewashed Dynabeads for 1 h. Depleted nuclear supernatant was incubated with 1 µg SNORD27 antisense oligonucleotide (2 ng/µl final) or random base oligonucleotide for 5 h followed by additional incubation for 1 h with preblocked Dynabeads and five washings with 1 mL ST2M+0.1NP40 buffer.

    Article Title: Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of theId3 Gene
    Article Snippet: DNA Affinity Binding Assay —The proteins translated by in vitro transcription/translation systems (Promega) were incubated with a double-stranded biotinylated DNA probe (1 mg) in gel-shift binding buffer ( ) containing 5 mg of poly(dI-dC), 20 mg of herring sperm DNA, and 0.5% Nonidet P-40 for 30 min on ice ( ). .. Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C. .. The DNA-bound proteins were analyzed by immunoblotting with the indicated antibodies.

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: In parallel, the 300 ng of each DNA library were pooled with the blocker #1, #2, and #3 reagents (Agilent), denatured for 5 min at 95°C, and then incubated at 65°C in a thermal cycler (MJ Research). .. We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen). .. The reaction product was then purified with the MinElute PCR purification kit (Qiagen) according to the manufacturer’s protocol.

    Article Title: Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells
    Article Snippet: The ECL counts of the surface-bound (acid-releasable) and intracellular (acid-resistant) biotinylated Fab for the transcytotic assay were determined as follow: 50 μl of the eluates and lysates were incubated with 4 μg/ml ruthenium- labeled affinity-purified goat anti–rat IgG F(ab′)2 fragment specific antibody for 2 h at RT. .. Thereafter, 1 μl of M-280 Streptavidin Dynabeads (Dynal, Hamburg, Germany) was added for 15 min at RT.

    Article Title: Recruitment of the SWI/SNF protein Brg1 by a multiprotein complex effects transcriptional repression in murine erythroid progenitors
    Article Snippet: Streptavidin-conjugated beads (M-280 Dynabeads, Dynal, Oslo, Norway) were washed three times with BW buffer {TE buffer [10 mM Tris/HCl (pH 8.0)/1 mM EDTA] containing 1 M NaCl} before use. .. A biotinylated, double-stranded oligonucleotide (25 pmol) corresponding to the E1G1 element in the P4.2 promoter and the previously described E box, GATA, and combination E box–GATA mutants [ ] were incubated with washed beads in 200 μl of BW buffer at room temperature for 20 min. DNA-loaded beads were washed twice with BW buffer and incubated with BW buffer containing 1% BSA at 4 °C overnight to block non-specific protein binding.

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol. .. For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: The biotinylated DNA was generated by PCR using M13-ssDNA as a DNA template. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100. .. The samples were run on 10% SDS–PAGE and analyzed by Western blotting.

    Affinity Binding Assay:

    Article Title: Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of theId3 Gene
    Article Snippet: DNA Affinity Binding Assay —The proteins translated by in vitro transcription/translation systems (Promega) were incubated with a double-stranded biotinylated DNA probe (1 mg) in gel-shift binding buffer ( ) containing 5 mg of poly(dI-dC), 20 mg of herring sperm DNA, and 0.5% Nonidet P-40 for 30 min on ice ( ). .. Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C.

    Expressing:

    Article Title: A tissue-specific landscape of sense/antisense transcription in the mouse intestine
    Article Snippet: Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment. .. First type of libraries (cDNA fragmented) was sequenced on a SOLiD V2 and V2.5 (replicates) sequencing by ligation sequencer following manufacturer's instructions, second type of libraries (RNA fragmented) on a SOLiD V4.

    Western Blot:

    Article Title: Recruitment of the SWI/SNF protein Brg1 by a multiprotein complex effects transcriptional repression in murine erythroid progenitors
    Article Snippet: Streptavidin-conjugated beads (M-280 Dynabeads, Dynal, Oslo, Norway) were washed three times with BW buffer {TE buffer [10 mM Tris/HCl (pH 8.0)/1 mM EDTA] containing 1 M NaCl} before use. .. Nuclear extract (200 μg) from undifferentiated MEL cells was then added to DNA-bound beads in binding buffer [20 mM Hepes, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM DTT (dithiothreitol) and 25% (v/v) glycerol] and incubated at room temperature for 30 min.

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    Transformation Assay:

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Amplicons were then ligated in to the Topo TA 2.1 PCR or Topo TA 4.0 sequencing vector and transformed into TOP10 electrocompetent cells (Invitrogen, Carlsbad, CA, USA). .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Allele-specific Oligonucleotide:

    Article Title: DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules
    Article Snippet: Target DNA (PCR products or up to 4 μg restricted genomic DNA) was added to a final volume of 20 μL 1 × DEB plus 0.38 μM bio-ASO and 1.5 μM competitor ASO in a 0.2-mL PCR tube (ABgene Thermo-Tube). .. Hybrids were captured by adding 0.18 mg (1.3 μL) Dynabeads M-280 Streptavidin (Dynal Biotech; pre-washed twice with 1 × DEB) and allowing binding to proceed for 10 min at A°C, with occasional gentle mixing to keep the beads suspended.

    Hybridization:

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: In parallel, the 300 ng of each DNA library were pooled with the blocker #1, #2, and #3 reagents (Agilent), denatured for 5 min at 95°C, and then incubated at 65°C in a thermal cycler (MJ Research). .. We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen). .. The reaction product was then purified with the MinElute PCR purification kit (Qiagen) according to the manufacturer’s protocol.

    Flow Cytometry:

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: Rpb1 was immunoprecipitated with 10 µg anti-RNA pol II (8WG16) mouse monoclonal antibodies coupled to 50 µl protein G–agarose beads (Roche). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. Immunoprecipitated chromatin was eluted by incubating two times for 10 min at 65°C in 10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 1% SDS.

    Article Title: Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells
    Article Snippet: Lipids were resuspended in 10 m m HEPES, pH 7.4, and 150 m m NaCl, frozen, and thawed 5 times using dry ice/acetone and a 40 °C water bath, extruded 19 times through two polycarbonate membranes with 100-nm pores (Avestin) using a LiposoFast-Basic (Avestin), and stored at 4 °C for use within 7 days. .. For flow cytometric assays, 1 μmol of lipids in the desired molar ratio were mixed with 10 nmol of N -(biotinyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanol-amine, triethylammonium (Invitrogen), and 6.4 nmol of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl- sn -glycero-3-phosphocholine (Invitrogen), chloroform was evaporated and lipids were resuspended in 25 m m HEPES and 150 m m NaCl, pH 7.7 (HBS), sonicated, and mixed with M-280 streptavidin-coated Dynabeads (Invitrogen) as previously described. .. To measure the kinetics of the DNA-C1q interaction, biotinylated DNA was coupled to a streptavidin-coated SA chip (GE Healthcare).

    Gas Chromatography:

    Article Title: DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules
    Article Snippet: Hybrids were captured by adding 0.18 mg (1.3 μL) Dynabeads M-280 Streptavidin (Dynal Biotech; pre-washed twice with 1 × DEB) and allowing binding to proceed for 10 min at A°C, with occasional gentle mixing to keep the beads suspended. .. Hybrids were captured by adding 0.18 mg (1.3 μL) Dynabeads M-280 Streptavidin (Dynal Biotech; pre-washed twice with 1 × DEB) and allowing binding to proceed for 10 min at A°C, with occasional gentle mixing to keep the beads suspended.

    Ligation:

    Article Title: A tissue-specific landscape of sense/antisense transcription in the mouse intestine
    Article Snippet: Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment. .. Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment.

    Protease Inhibitor:

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
    Article Snippet: Pulldown was performed with RNA oligonucleotide [2′- O -methylated phosphorothioate with tetra-ethyleneglycol biotin (TEG-Biotin) on 3′-end], having 20 nucleotides complementary to the first antisense box of SNORD27 RNA. .. M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension. .. Nuclear supernatant corresponding to five 15-cm Petri dishes of the HeLa cells in ST2M+0.1NP40 buffer was depleted from unspecific streptavidin binding material by incubation with prewashed Dynabeads for 1 h. Depleted nuclear supernatant was incubated with 1 µg SNORD27 antisense oligonucleotide (2 ng/µl final) or random base oligonucleotide for 5 h followed by additional incubation for 1 h with preblocked Dynabeads and five washings with 1 mL ST2M+0.1NP40 buffer.

    SDS Page:

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
    Article Snippet: M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension. .. Nuclear supernatant corresponding to five 15-cm Petri dishes of the HeLa cells in ST2M+0.1NP40 buffer was depleted from unspecific streptavidin binding material by incubation with prewashed Dynabeads for 1 h. Depleted nuclear supernatant was incubated with 1 µg SNORD27 antisense oligonucleotide (2 ng/µl final) or random base oligonucleotide for 5 h followed by additional incubation for 1 h with preblocked Dynabeads and five washings with 1 mL ST2M+0.1NP40 buffer.

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    Generated:

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: The Applied Biosystem 3730 XL capillary sequencer (Applied Biosystems, Foster City, CA, USA) performed all sequencing, which included both forward and reverse reads generated with M13 primers and ABI BigDye 3.1 chemistry. .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: Biotinylated promoter DNA fragments were generated by PCR using a 5′-biotinylated sense oligonucleotide. .. For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: The biotinylated DNA was generated by PCR using M13-ssDNA as a DNA template. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    other:

    Article Title: Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments
    Article Snippet: Dynabeads M-280 Streptavidin + the pull-down fractions were then washed three times with 200 μL EB-EDTA buffer and eventually resuspended with 30 μL of 1X denaturing loading buffer.

    Protein Concentration:

    Article Title: The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage
    Article Snippet: Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose. .. Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins
    Article Snippet: One microgram of paramagnetic polystyrene microspheres covalently linked to streptavidin (Dynabeads M-280 Streptavidin; Dynal, Great Neck, NY) was washed twice in diethyl pyrocarbonate-treated solution A (0.1 M NaOH/0.05 M NaCl) and once in diethyl pyrocarbonate-treated solution B (0.1 M NaOH) by placing the tube in a magnetic particle concentrator for 3 min and then removing the free fraction, to ensure an RNase-free environment. .. The 16A11 mAb–beads complex was washed five times in PBS + 0.1% BSA and, after removal from the concentrator, incubated for 2 h with different aliquots of mouse total hippocampal tissue lysate, with continuous gentle mixing.

    Sonication:

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions. .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions.

    Article Title: Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells
    Article Snippet: Lipids were resuspended in 10 m m HEPES, pH 7.4, and 150 m m NaCl, frozen, and thawed 5 times using dry ice/acetone and a 40 °C water bath, extruded 19 times through two polycarbonate membranes with 100-nm pores (Avestin) using a LiposoFast-Basic (Avestin), and stored at 4 °C for use within 7 days. .. For flow cytometric assays, 1 μmol of lipids in the desired molar ratio were mixed with 10 nmol of N -(biotinyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanol-amine, triethylammonium (Invitrogen), and 6.4 nmol of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl- sn -glycero-3-phosphocholine (Invitrogen), chloroform was evaporated and lipids were resuspended in 25 m m HEPES and 150 m m NaCl, pH 7.7 (HBS), sonicated, and mixed with M-280 streptavidin-coated Dynabeads (Invitrogen) as previously described. .. To measure the kinetics of the DNA-C1q interaction, biotinylated DNA was coupled to a streptavidin-coated SA chip (GE Healthcare).

    Affinity Purification:

    Article Title: Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells
    Article Snippet: The ECL counts of the surface-bound (acid-releasable) and intracellular (acid-resistant) biotinylated Fab for the transcytotic assay were determined as follow: 50 μl of the eluates and lysates were incubated with 4 μg/ml ruthenium- labeled affinity-purified goat anti–rat IgG F(ab′)2 fragment specific antibody for 2 h at RT. .. Thereafter, 1 μl of M-280 Streptavidin Dynabeads (Dynal, Hamburg, Germany) was added for 15 min at RT.

    Binding Assay:

    Article Title: DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules
    Article Snippet: DNA was denatured at 96°C for 75 sec on a GeneAmp PCR System 9700 thermal cycler (PE Applied Biosystems), then step-down annealed from (A+9)°C to (A+1)°C over 3 min (where A°C is the optimal annealing temperature determined empirically), and held at A°C for 2 min. DNA was then transferred to a siliconized Eppendorf tube (Treff Lab polypropylene tube siliconized with dimethyldichlorosilane solution, BDH; siliconization is essential to prevent nonspecific recovery of single-stranded DNA) and held at A°C. .. Hybrids were captured by adding 0.18 mg (1.3 μL) Dynabeads M-280 Streptavidin (Dynal Biotech; pre-washed twice with 1 × DEB) and allowing binding to proceed for 10 min at A°C, with occasional gentle mixing to keep the beads suspended. .. Beads were immediately separated at room temperature ( < 30 sec) using a Dynal MPC-E magnetic particle concentrator, and unbound DNA transferred to a 0.2-mL PCR tube.

    Article Title: Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins
    Article Snippet: Paragraph title: In Vivo RNA–Protein Binding. ... One microgram of paramagnetic polystyrene microspheres covalently linked to streptavidin (Dynabeads M-280 Streptavidin; Dynal, Great Neck, NY) was washed twice in diethyl pyrocarbonate-treated solution A (0.1 M NaOH/0.05 M NaCl) and once in diethyl pyrocarbonate-treated solution B (0.1 M NaOH) by placing the tube in a magnetic particle concentrator for 3 min and then removing the free fraction, to ensure an RNase-free environment.

    Article Title: Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of theId3 Gene
    Article Snippet: DNA Affinity Binding Assay —The proteins translated by in vitro transcription/translation systems (Promega) were incubated with a double-stranded biotinylated DNA probe (1 mg) in gel-shift binding buffer ( ) containing 5 mg of poly(dI-dC), 20 mg of herring sperm DNA, and 0.5% Nonidet P-40 for 30 min on ice ( ). .. Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C.

    Article Title: Recruitment of the SWI/SNF protein Brg1 by a multiprotein complex effects transcriptional repression in murine erythroid progenitors
    Article Snippet: Streptavidin-conjugated beads (M-280 Dynabeads, Dynal, Oslo, Norway) were washed three times with BW buffer {TE buffer [10 mM Tris/HCl (pH 8.0)/1 mM EDTA] containing 1 M NaCl} before use. .. A biotinylated, double-stranded oligonucleotide (25 pmol) corresponding to the E1G1 element in the P4.2 promoter and the previously described E box, GATA, and combination E box–GATA mutants [ ] were incubated with washed beads in 200 μl of BW buffer at room temperature for 20 min. DNA-loaded beads were washed twice with BW buffer and incubated with BW buffer containing 1% BSA at 4 °C overnight to block non-specific protein binding.

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol. .. Consistently, we observed a DNA binding efficiency of > 90% (data not shown).

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: Paragraph title: Chromatin binding assay ... Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    In Vivo:

    Article Title: Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins
    Article Snippet: Paragraph title: In Vivo RNA–Protein Binding. ... One microgram of paramagnetic polystyrene microspheres covalently linked to streptavidin (Dynabeads M-280 Streptavidin; Dynal, Great Neck, NY) was washed twice in diethyl pyrocarbonate-treated solution A (0.1 M NaOH/0.05 M NaCl) and once in diethyl pyrocarbonate-treated solution B (0.1 M NaOH) by placing the tube in a magnetic particle concentrator for 3 min and then removing the free fraction, to ensure an RNase-free environment.

    RNA Sequencing Assay:

    Article Title: A tissue-specific landscape of sense/antisense transcription in the mouse intestine
    Article Snippet: Paragraph title: RNA-Seq ... Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment.

    Pull Down Assay:

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: Paragraph title: Promoter pull-down assay ... For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Methylation:

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
    Article Snippet: Pulldown was performed with RNA oligonucleotide [2′- O -methylated phosphorothioate with tetra-ethyleneglycol biotin (TEG-Biotin) on 3′-end], having 20 nucleotides complementary to the first antisense box of SNORD27 RNA. .. M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension.

    Isolation:

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Total RNA was isolated from clonal cultures of A. ricciae , P. acuticornis , P. roseola , H. rosa , and B. manjavacas using RNAqueous Micro Kit (Ambion). .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: Protein–DNA complexes were isolated by immunoprecipitation for 1 h and 45 min at room temperature from 200 µl of chromatin extract. .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: Chromatin was isolated through a 30% sucrose solution in CI buffer at 6000 g for 30 min at 4°C. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    Polymerase Chain Reaction:

    Article Title: Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries
    Article Snippet: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7; Ambion, Cat. No AM9861) NaCl (NaCl powder, Fisher Scientific, Cat. No. S271-10) Tris (Trizma® base, Sigma-Aldrich, Cat.No. .. T1503-1KG) EDTA (EDTA powder, Sigma-Aldrich, Cat.No. s657-500) RNase A (Qiagen, Cat. No. 19101) QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) Platinum Pfx (Invitrogen, Cat. No. 11708-021) 10mM dNTPs (Invitrogen, Cat. No. 18427-013) Dynabeads M-280 Streptavidin (Invitrogen, Cat. No. 112-05D) Hexanucleotide Mix (Roche, Cat. No. 11277081001) Klenow Fragment (3’ - 5’ exo-; 5 U/μL) (New England Biolabs, Cat. No. M0212S) MmeI (New England Biolabs, Cat. No. R0637S) T4 DNA ligase (2,000,000 U/mL) (New England Biolabs, Cat. No. M0202T) Agarose (Roche, Cat. No. 11388991-001) Electrophoresis buffer (TBE, Borate Buffer , or equivalent) 250mM NaOH (Fisher Scientific, Cat. No. S318 3) Boric Acid (Sigma-Aldrich, Cat. No. B7901-500) 10bp DNA Ladder (Invitrogen, Cat. No. 10821-015) GelGreen Nucleic Acid Stain, 10,000x stock prepared in water (Biotium, Cat. No. 41005) 20% Ficoll 400 (Sigma-Aldrich, Cat. No. F4375) 0.1M disodium EDTA (Sigma-Aldrich, Cat. No. BP120-500) 0.25% xylene cyanol (Sigma-Aldrich, Cat. No. X4126-10G) 0.25% bromophenol blue; (Sigma-Aldrich, Cat. No. 114391-5G) QIAquick Gel Extractio Kit (Qiagen, Cat. No. 28706) Primer sequences are provided in . .. Primers were obtained from Integrated DNA Technologies (IDT).

    Article Title: Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX
    Article Snippet: Taq PCR MasterMix and 50 bp DNA Ladder were purchased from Tiangen (Tiangen Biotech Co., Ltd., Beijing, China). .. Dynabeads M-280 Streptavidin was purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Amplicons were then ligated in to the Topo TA 2.1 PCR or Topo TA 4.0 sequencing vector and transformed into TOP10 electrocompetent cells (Invitrogen, Carlsbad, CA, USA). .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: A tissue-specific landscape of sense/antisense transcription in the mouse intestine
    Article Snippet: For second strand synthesis and amplification, a 5'-biotinylated version of PCR primer II was employed. .. Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment.

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Article Title: DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules
    Article Snippet: DNA was denatured at 96°C for 75 sec on a GeneAmp PCR System 9700 thermal cycler (PE Applied Biosystems), then step-down annealed from (A+9)°C to (A+1)°C over 3 min (where A°C is the optimal annealing temperature determined empirically), and held at A°C for 2 min. DNA was then transferred to a siliconized Eppendorf tube (Treff Lab polypropylene tube siliconized with dimethyldichlorosilane solution, BDH; siliconization is essential to prevent nonspecific recovery of single-stranded DNA) and held at A°C. .. Hybrids were captured by adding 0.18 mg (1.3 μL) Dynabeads M-280 Streptavidin (Dynal Biotech; pre-washed twice with 1 × DEB) and allowing binding to proceed for 10 min at A°C, with occasional gentle mixing to keep the beads suspended.

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Wear protective clothing and gloves and handle under a fume hood. .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. SpectraMax M2 multimode microplate reader (Molecular Devices) AFA S2 (Covaris) DynaMag-2 (Invitrogen, cat. no. 123-21D) NanoDrop 1000 (Thermo Scientific) Thermocycler (Applied Biosystems, 2720) Nutator (Southwest Science, SB3D2300) Desktop centrifuge (Eppendorf, 5424)

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: The reaction product was purified using a QIAquick PCR purification kit (Qiagen) and eluted into 20 μl EB. .. We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen).

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: Templates were synthesized by PCR from pBSAd20 or pBSAd22 using biotinylated, upstream primer Ade3 (5′-biotin-GTTGGGTAACGCCAGGG-3′) and downstream primer T3-L (5′-GCGCAATTAACCCTCACTAAA-3′). .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions.

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: Biotinylated promoter DNA fragments were generated by PCR using a 5′-biotinylated sense oligonucleotide. .. For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: The biotinylated DNA was generated by PCR using M13-ssDNA as a DNA template. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    Size-exclusion Chromatography:

    Article Title: DNA Enrichment by Allele-Specific Hybridization (DEASH): A Novel Method for Haplotyping and for Detecting Low-Frequency Base Substitutional Variants and Recombinant DNA Molecules
    Article Snippet: DNA was denatured at 96°C for 75 sec on a GeneAmp PCR System 9700 thermal cycler (PE Applied Biosystems), then step-down annealed from (A+9)°C to (A+1)°C over 3 min (where A°C is the optimal annealing temperature determined empirically), and held at A°C for 2 min. DNA was then transferred to a siliconized Eppendorf tube (Treff Lab polypropylene tube siliconized with dimethyldichlorosilane solution, BDH; siliconization is essential to prevent nonspecific recovery of single-stranded DNA) and held at A°C. .. Hybrids were captured by adding 0.18 mg (1.3 μL) Dynabeads M-280 Streptavidin (Dynal Biotech; pre-washed twice with 1 × DEB) and allowing binding to proceed for 10 min at A°C, with occasional gentle mixing to keep the beads suspended.

    Electrophoretic Mobility Shift Assay:

    Article Title: Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of theId3 Gene
    Article Snippet: DNA Affinity Binding Assay —The proteins translated by in vitro transcription/translation systems (Promega) were incubated with a double-stranded biotinylated DNA probe (1 mg) in gel-shift binding buffer ( ) containing 5 mg of poly(dI-dC), 20 mg of herring sperm DNA, and 0.5% Nonidet P-40 for 30 min on ice ( ). .. Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C.

    Purification:

    Article Title: Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries
    Article Snippet: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7; Ambion, Cat. No AM9861) NaCl (NaCl powder, Fisher Scientific, Cat. No. S271-10) Tris (Trizma® base, Sigma-Aldrich, Cat.No. .. T1503-1KG) EDTA (EDTA powder, Sigma-Aldrich, Cat.No. s657-500) RNase A (Qiagen, Cat. No. 19101) QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) Platinum Pfx (Invitrogen, Cat. No. 11708-021) 10mM dNTPs (Invitrogen, Cat. No. 18427-013) Dynabeads M-280 Streptavidin (Invitrogen, Cat. No. 112-05D) Hexanucleotide Mix (Roche, Cat. No. 11277081001) Klenow Fragment (3’ - 5’ exo-; 5 U/μL) (New England Biolabs, Cat. No. M0212S) MmeI (New England Biolabs, Cat. No. R0637S) T4 DNA ligase (2,000,000 U/mL) (New England Biolabs, Cat. No. M0202T) Agarose (Roche, Cat. No. 11388991-001) Electrophoresis buffer (TBE, Borate Buffer , or equivalent) 250mM NaOH (Fisher Scientific, Cat. No. S318 3) Boric Acid (Sigma-Aldrich, Cat. No. B7901-500) 10bp DNA Ladder (Invitrogen, Cat. No. 10821-015) GelGreen Nucleic Acid Stain, 10,000x stock prepared in water (Biotium, Cat. No. 41005) 20% Ficoll 400 (Sigma-Aldrich, Cat. No. F4375) 0.1M disodium EDTA (Sigma-Aldrich, Cat. No. BP120-500) 0.25% xylene cyanol (Sigma-Aldrich, Cat. No. X4126-10G) 0.25% bromophenol blue; (Sigma-Aldrich, Cat. No. 114391-5G) QIAquick Gel Extractio Kit (Qiagen, Cat. No. 28706) Primer sequences are provided in . .. Primers were obtained from Integrated DNA Technologies (IDT).

    Article Title: An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair
    Article Snippet: The DNA was purified on a Qiagen column and then treated with 300 μ m N -acetoxy-2-acetylaminofluorene (AAAF) or ethanol (for unmodified DNA) for 3 h at room temperature in the dark. .. To immobilize the DNA, 300 μl of Dynabeads M-280 streptavidin (Invitrogen) were prewashed in PBS containing 1 mg/ml BSA and 0.05% Nonidet P-40 and then incubated with 15 μg of biotinylated DNA in 10 m m Tris, 1 m m EDTA, 1 m NaCl.

    Article Title: Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX
    Article Snippet: The purified AFP protein (100 μg/ml) was purchased from Fitzgerald (Fitzgerald, MA, USA). .. Dynabeads M-280 Streptavidin was purchased from Invitrogen (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Colonies were grown in 96-well plates and the Biomek FX liquid handling robot (Beckman Coulter, Fullerton, CA, USA) purified positive plasmids using a standard alkaline lysis protocol. .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Wear protective clothing and gloves and handle under a fume hood. .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. SpectraMax M2 multimode microplate reader (Molecular Devices) AFA S2 (Covaris) DynaMag-2 (Invitrogen, cat. no. 123-21D) NanoDrop 1000 (Thermo Scientific) Thermocycler (Applied Biosystems, 2720) Nutator (Southwest Science, SB3D2300) Desktop centrifuge (Eppendorf, 5424)

    Article Title: Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins
    Article Snippet: One microgram of paramagnetic polystyrene microspheres covalently linked to streptavidin (Dynabeads M-280 Streptavidin; Dynal, Great Neck, NY) was washed twice in diethyl pyrocarbonate-treated solution A (0.1 M NaOH/0.05 M NaCl) and once in diethyl pyrocarbonate-treated solution B (0.1 M NaOH) by placing the tube in a magnetic particle concentrator for 3 min and then removing the free fraction, to ensure an RNase-free environment. .. After removal of the free fraction in the magnetic concentrator, beads were washed three times in PBS + 0.1% BSA, and total RNA was extracted from both fractions and subjected to qualitative RT-PCR.

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
    Article Snippet: Paragraph title: Purification of RNA and Bound Proteins Using Biotinylated Antisense Oligonucleotides. ... M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension.

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: The reaction product was purified using a QIAquick PCR purification kit (Qiagen) and eluted into 20 μl EB. .. We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen).

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: Templates were synthesized by PCR from pBSAd20 or pBSAd22 using biotinylated, upstream primer Ade3 (5′-biotin-GTTGGGTAACGCCAGGG-3′) and downstream primer T3-L (5′-GCGCAATTAACCCTCACTAAA-3′). .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions. .. The immobilized templates were then blocked with 1% BSA in BC-100 buffer (20 mM HEPES–KOH (pH 7.6), 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 250 μM PMSF) for 20 min at RT.

    Sequencing:

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Synthesis of cDNA with first strand primer 5′ CTA GAG GCC GAG GCG GCC GAT TTT TTT TTT TTT TTT TTT UVN 3′ made use of the template switching property of Superscript II (Invitrogen) to incorporate barcoded, biotintylated 5′ adapters that matched the “A” sequence primers used in 454 FLX pyrosequencing (5′ GCC TCC CTC GCG CCA TCA Gxx xxx GG, where xxxxx is CACTG for B. manjavacas ). .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: A tissue-specific landscape of sense/antisense transcription in the mouse intestine
    Article Snippet: Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment. .. Subsequently, SOLiD V2 fragment library protocol (Applied Biosystems) was applied and transcript ends were depleted by two rounds of Dynabeads M-280 streptavidin (Invitrogen) treatment.

    Article Title: Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of theId3 Gene
    Article Snippet: Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C. .. Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C.

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: Paragraph title: Chip capture library preparation, hybridization and sequencing ... We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen).

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: The pBSAd22 plasmid contains, in addition, a 399-nt sequence including the adenovirus major late promoter (AdMLP) immediately downstream of the T7 promoter. .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions.

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: For the generation of fragments GPEET –246/–162, RRNA –257/–162 and rUSE1-mut, the 5′-biotinylated T7 sense oligonucleotide was used which added 41 bp of vector sequence 5′ to the promoter region, whereas for fragments SLRNA –126/–18 and USE1-mut, the biotinylated oligonucleotide SL14 was used which is sense to SLRNA promoter positions 126 to 107 and did not add extra base pairs to the fragment. .. For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Labeling:

    Article Title: Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells
    Article Snippet: The ECL counts of the surface-bound (acid-releasable) and intracellular (acid-resistant) biotinylated Fab for the transcytotic assay were determined as follow: 50 μl of the eluates and lysates were incubated with 4 μg/ml ruthenium- labeled affinity-purified goat anti–rat IgG F(ab′)2 fragment specific antibody for 2 h at RT. .. Thereafter, 1 μl of M-280 Streptavidin Dynabeads (Dynal, Hamburg, Germany) was added for 15 min at RT.

    Bradford Protein Assay:

    Article Title: The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage
    Article Snippet: Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose. .. Immunoprecipitations were done using Anti-Flag M2 affinity gel (Sigma, A2220), Monoclonal anti-HA agarose (Sigma, A2095), Dynabeads M-280 Streptavidin (Invitrogen, 112.05D) or Treacle antibody (Sigma Life Science, HPA038237), in combination with Protein A agarose.

    Staining:

    Article Title: Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries
    Article Snippet: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7; Ambion, Cat. No AM9861) NaCl (NaCl powder, Fisher Scientific, Cat. No. S271-10) Tris (Trizma® base, Sigma-Aldrich, Cat.No. .. T1503-1KG) EDTA (EDTA powder, Sigma-Aldrich, Cat.No. s657-500) RNase A (Qiagen, Cat. No. 19101) QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) Platinum Pfx (Invitrogen, Cat. No. 11708-021) 10mM dNTPs (Invitrogen, Cat. No. 18427-013) Dynabeads M-280 Streptavidin (Invitrogen, Cat. No. 112-05D) Hexanucleotide Mix (Roche, Cat. No. 11277081001) Klenow Fragment (3’ - 5’ exo-; 5 U/μL) (New England Biolabs, Cat. No. M0212S) MmeI (New England Biolabs, Cat. No. R0637S) T4 DNA ligase (2,000,000 U/mL) (New England Biolabs, Cat. No. M0202T) Agarose (Roche, Cat. No. 11388991-001) Electrophoresis buffer (TBE, Borate Buffer , or equivalent) 250mM NaOH (Fisher Scientific, Cat. No. S318 3) Boric Acid (Sigma-Aldrich, Cat. No. B7901-500) 10bp DNA Ladder (Invitrogen, Cat. No. 10821-015) GelGreen Nucleic Acid Stain, 10,000x stock prepared in water (Biotium, Cat. No. 41005) 20% Ficoll 400 (Sigma-Aldrich, Cat. No. F4375) 0.1M disodium EDTA (Sigma-Aldrich, Cat. No. BP120-500) 0.25% xylene cyanol (Sigma-Aldrich, Cat. No. X4126-10G) 0.25% bromophenol blue; (Sigma-Aldrich, Cat. No. 114391-5G) QIAquick Gel Extractio Kit (Qiagen, Cat. No. 28706) Primer sequences are provided in . .. Primers were obtained from Integrated DNA Technologies (IDT).

    Article Title: Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
    Article Snippet: M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension. .. M-280 Streptavidin Dynabeads (Life Technology) were washed three times with 10 vol ST2M+0.1NP40 buffer [0.1 M NaCl, 10 mM Tris⋅HCl, pH 8.0, 2 mM MgCl2 , 0.1% Nonidet P-40, complete protease inhibitor mixture (Life Technology), 1 µg/µL RNase Inhibitor (New England Biolabs)] and preblocked by incubation with 500 ng/µL yeast tRNA and 1 mg/mL BSA in ST2M+0.1NP40 buffer for 1 h. Each sample was incubated with an aliquot of beads corresponding to 30 µL initial suspension.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries
    Article Snippet: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7; Ambion, Cat. No AM9861) NaCl (NaCl powder, Fisher Scientific, Cat. No. S271-10) Tris (Trizma® base, Sigma-Aldrich, Cat.No. .. T1503-1KG) EDTA (EDTA powder, Sigma-Aldrich, Cat.No. s657-500) RNase A (Qiagen, Cat. No. 19101) QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) Platinum Pfx (Invitrogen, Cat. No. 11708-021) 10mM dNTPs (Invitrogen, Cat. No. 18427-013) Dynabeads M-280 Streptavidin (Invitrogen, Cat. No. 112-05D) Hexanucleotide Mix (Roche, Cat. No. 11277081001) Klenow Fragment (3’ - 5’ exo-; 5 U/μL) (New England Biolabs, Cat. No. M0212S) MmeI (New England Biolabs, Cat. No. R0637S) T4 DNA ligase (2,000,000 U/mL) (New England Biolabs, Cat. No. M0202T) Agarose (Roche, Cat. No. 11388991-001) Electrophoresis buffer (TBE, Borate Buffer , or equivalent) 250mM NaOH (Fisher Scientific, Cat. No. S318 3) Boric Acid (Sigma-Aldrich, Cat. No. B7901-500) 10bp DNA Ladder (Invitrogen, Cat. No. 10821-015) GelGreen Nucleic Acid Stain, 10,000x stock prepared in water (Biotium, Cat. No. 41005) 20% Ficoll 400 (Sigma-Aldrich, Cat. No. F4375) 0.1M disodium EDTA (Sigma-Aldrich, Cat. No. BP120-500) 0.25% xylene cyanol (Sigma-Aldrich, Cat. No. X4126-10G) 0.25% bromophenol blue; (Sigma-Aldrich, Cat. No. 114391-5G) QIAquick Gel Extractio Kit (Qiagen, Cat. No. 28706) Primer sequences are provided in . .. Primers were obtained from Integrated DNA Technologies (IDT).

    Real-time Polymerase Chain Reaction:

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Chromatin Immunoprecipitation:

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: Paragraph title: Chromatin immunoprecipitation ... TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    Article Title: Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.)
    Article Snippet: Paragraph title: Chip capture library preparation, hybridization and sequencing ... We then mixed 12 μl of hybridization solution, 5 μl of mixed SureSelect Oligo Capture Library, 11 μl of the DNA library, 1 μl H2 O, and 1 μl RNase block (Agilent), incubated for 24 hours at 65°C in a thermal cycler (MJ Research) and captured with the Streptavidin M-280 Dynabeads (Invitrogen).

    SPR Assay:

    Article Title: Annexin A2 and A5 Serve as New Ligands for C1q on Apoptotic Cells
    Article Snippet: For flow cytometric assays, 1 μmol of lipids in the desired molar ratio were mixed with 10 nmol of N -(biotinyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanol-amine, triethylammonium (Invitrogen), and 6.4 nmol of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl- sn -glycero-3-phosphocholine (Invitrogen), chloroform was evaporated and lipids were resuspended in 25 m m HEPES and 150 m m NaCl, pH 7.7 (HBS), sonicated, and mixed with M-280 streptavidin-coated Dynabeads (Invitrogen) as previously described. .. For flow cytometric assays, 1 μmol of lipids in the desired molar ratio were mixed with 10 nmol of N -(biotinyl)-1,2-dihexadecanoyl- sn -glycero-3-phosphoethanol-amine, triethylammonium (Invitrogen), and 6.4 nmol of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl- sn -glycero-3-phosphocholine (Invitrogen), chloroform was evaporated and lipids were resuspended in 25 m m HEPES and 150 m m NaCl, pH 7.7 (HBS), sonicated, and mixed with M-280 streptavidin-coated Dynabeads (Invitrogen) as previously described.

    Plasmid Preparation:

    Article Title: An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair
    Article Snippet: The AAAF treatment protocol yields ∼1 AAAF-guanine adduct every 200–250 bp of DNA, resulting in 10–14 lesions per plasmid ( ). .. To immobilize the DNA, 300 μl of Dynabeads M-280 streptavidin (Invitrogen) were prewashed in PBS containing 1 mg/ml BSA and 0.05% Nonidet P-40 and then incubated with 15 μg of biotinylated DNA in 10 m m Tris, 1 m m EDTA, 1 m NaCl.

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Amplicons were then ligated in to the Topo TA 2.1 PCR or Topo TA 4.0 sequencing vector and transformed into TOP10 electrocompetent cells (Invitrogen, Carlsbad, CA, USA). .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Article Title: p54nrb associates with the 5? splice site within large transcription/splicing complexes
    Article Snippet: The pBSAd22 plasmid contains, in addition, a 399-nt sequence including the adenovirus major late promoter (AdMLP) immediately downstream of the T7 promoter. .. The resulting fragments were gel purified and bound to M-280 streptavidin Dynabeads (Dynal) according to the manufacturer's instructions.

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: For the generation of fragments GPEET –246/–162, RRNA –257/–162 and rUSE1-mut, the 5′-biotinylated T7 sense oligonucleotide was used which added 41 bp of vector sequence 5′ to the promoter region, whereas for fragments SLRNA –126/–18 and USE1-mut, the biotinylated oligonucleotide SL14 was used which is sense to SLRNA promoter positions 126 to 107 and did not add extra base pairs to the fragment. .. For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Electrophoresis:

    Article Title: Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries
    Article Snippet: TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7; Ambion, Cat. No AM9861) NaCl (NaCl powder, Fisher Scientific, Cat. No. S271-10) Tris (Trizma® base, Sigma-Aldrich, Cat.No. .. T1503-1KG) EDTA (EDTA powder, Sigma-Aldrich, Cat.No. s657-500) RNase A (Qiagen, Cat. No. 19101) QIAquick PCR Purification Kit (Qiagen, Cat. No. 28106) Platinum Pfx (Invitrogen, Cat. No. 11708-021) 10mM dNTPs (Invitrogen, Cat. No. 18427-013) Dynabeads M-280 Streptavidin (Invitrogen, Cat. No. 112-05D) Hexanucleotide Mix (Roche, Cat. No. 11277081001) Klenow Fragment (3’ - 5’ exo-; 5 U/μL) (New England Biolabs, Cat. No. M0212S) MmeI (New England Biolabs, Cat. No. R0637S) T4 DNA ligase (2,000,000 U/mL) (New England Biolabs, Cat. No. M0202T) Agarose (Roche, Cat. No. 11388991-001) Electrophoresis buffer (TBE, Borate Buffer , or equivalent) 250mM NaOH (Fisher Scientific, Cat. No. S318 3) Boric Acid (Sigma-Aldrich, Cat. No. B7901-500) 10bp DNA Ladder (Invitrogen, Cat. No. 10821-015) GelGreen Nucleic Acid Stain, 10,000x stock prepared in water (Biotium, Cat. No. 41005) 20% Ficoll 400 (Sigma-Aldrich, Cat. No. F4375) 0.1M disodium EDTA (Sigma-Aldrich, Cat. No. BP120-500) 0.25% xylene cyanol (Sigma-Aldrich, Cat. No. X4126-10G) 0.25% bromophenol blue; (Sigma-Aldrich, Cat. No. 114391-5G) QIAquick Gel Extractio Kit (Qiagen, Cat. No. 28706) Primer sequences are provided in . .. Primers were obtained from Integrated DNA Technologies (IDT).

    Recombinant:

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: For DNA binding assay, recombinant MCM proteins were incubated at 22°C for 20 min in MCM-depleted extracts supplemented with a biotinylated 1 kb DNA fragment that was prebound to streptavidin beads. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    In Vitro:

    Article Title: Rho/Rho-associated Kinase Signal Regulates Myogenic Differentiation via Myocardin-related Transcription Factor-A/Smad-dependent Transcription of theId3 Gene
    Article Snippet: DNA Affinity Binding Assay —The proteins translated by in vitro transcription/translation systems (Promega) were incubated with a double-stranded biotinylated DNA probe (1 mg) in gel-shift binding buffer ( ) containing 5 mg of poly(dI-dC), 20 mg of herring sperm DNA, and 0.5% Nonidet P-40 for 30 min on ice ( ). .. Streptavidin M-280 Dynabeads (Dynal) were then added, and the mixture was further incubated with rotation for 2 h at 4 °C.

    Article Title: The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements
    Article Snippet: For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol. .. For each reaction, 500 ng of biotinylated DNA fragments were coupled to 10 µl (100 µg) of RNase-free, paramagnetic M-280 Streptavidin Dynabeads (Dynal) according to the manufacturer’s protocol.

    Immunoprecipitation:

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: Rpb1 was immunoprecipitated with 10 µg anti-RNA pol II (8WG16) mouse monoclonal antibodies coupled to 50 µl protein G–agarose beads (Roche). .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal). .. Immunoprecipitated chromatin was eluted by incubating two times for 10 min at 65°C in 10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 1% SDS.

    Alkaline Lysis:

    Article Title: A Mitogenomic Re-Evaluation of the Bdelloid Phylogeny and Relationships among the Syndermata
    Article Snippet: Colonies were grown in 96-well plates and the Biomek FX liquid handling robot (Beckman Coulter, Fullerton, CA, USA) purified positive plasmids using a standard alkaline lysis protocol. .. To prepare libraries for pyrosequencing, 3–5 µg of cDNA was sheared using an Aeromist Nebulizer (Allied Healthcare Products) for 3–4 min at 50 psi N2 , and the biotintylated 5′ EST ends of the fragmented library were captured with Dynabeads M-280 Streptavidin (Invitrogen).

    Lysis:

    Article Title: The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation
    Article Snippet: One part was washed twice with FA lysis buffer containing 0.5 M NaCl, twice in 10 mM Tris–HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.5% Na-deoxycholate, and once in 10 mM Tris–HCl, pH 8.0 and 1 mM EDTA. .. TAP-tagged and Avitag-tagged proteins were immunoprecipitated with 50 µl immunoglobulin G Sepharose 6 fast flow beads (Amersham) or 60 µl Dynabeads M-280 Streptavidin (Dynal).

    DNA Binding Assay:

    Article Title: The ATPase activity of MCM2-7 is dispensable for pre-RC assembly but is required for DNA unwinding
    Article Snippet: For DNA binding assay, recombinant MCM proteins were incubated at 22°C for 20 min in MCM-depleted extracts supplemented with a biotinylated 1 kb DNA fragment that was prebound to streptavidin beads. .. Following incubation, the DNA-bound streptavidin beads (Dynabeads M-280 Streptavidin from Dynal Biotech) were washed in XB buffer+0.2% Triton X-100.

    Gel Extraction:

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Wear protective clothing and gloves and handle under a fume hood. .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. SpectraMax M2 multimode microplate reader (Molecular Devices) AFA S2 (Covaris) DynaMag-2 (Invitrogen, cat. no. 123-21D) NanoDrop 1000 (Thermo Scientific) Thermocycler (Applied Biosystems, 2720) Nutator (Southwest Science, SB3D2300) Desktop centrifuge (Eppendorf, 5424)

    Hood:

    Article Title: The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine
    Article Snippet: Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500) .. Sodium acetate (Sigma-Aldrich, cat. no. S2889-250G) QIAquick PCR purification kit (Qiagen, cat. no. 28106) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Illustra MicroSpin G-50 columns (GE Healthcare, cat. no. 27-5330-01) Dynabeads M280-Streptavidin (Invitrogen, cat. no. 11206D) QuantIt OliGreen kit (Invitrogen, cat. no. ) PBS, 10× (Meditech CellGro, cat. no. 46-013-CM) Deoxynucleotide solution set (New England Biolabs, cat. no. N0446S) Hydroxymethyl dCTP (Bioline, cat. no. BIO-39046) pUC19 DNA (New England Biolabs, cat. no. N3041S) T4 phage β-glucosyltransferase (T4-BGT; New England Biolabs, cat. no. M0357S) AhdI (New England Biolabs, cat. no. R0584S) FspI (New England Biolabs, cat. no. R0135S) HindIII (New England Biolabs, cat. no. R0104S) NdeI (New England Biolabs, cat. no. R0111S) DNA polymerase, Klenow fragment (New England Biolabs, cat. no. M0210S) Ethanol (Sigma-Aldrich, cat. no. E7023) Tris (Sigma-Aldrich, cat. no. 154563) Agarose (Invitrogen, cat. no. 16500500)

    Electrochemiluminescence:

    Article Title: Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells
    Article Snippet: Paragraph title: Electrochemiluminescence Detection System ... Thereafter, 1 μl of M-280 Streptavidin Dynabeads (Dynal, Hamburg, Germany) was added for 15 min at RT.

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    Thermo Fisher streptavidin coated beads
    GSK3α/β binds and phosphorylates Arc protein. (A) GSK3β directly associates with Arc protein. Purified GST-Arc protein of rat origin was incubated with protein lysate from rat hippocampus. GSK3α/β binding was determined by Western blot. (B) GSK3α/β may directly associate with Arc protein in cells. Rat and human Arc or β-Gal proteins that were overexpressed in HEK293 cells were captured on <t>streptavidin-coated</t> beads and incubated with mouse hippocampal lysate. Bait-kinase binding was estimated by probing the blot with anti-GSK3α/β antibody. Bait (e.g., biotinylated Arc or β-Gal) was detected using fluorescently labeled streptavidin. (C) Kinase dead (KD) mutants of GSK3α (K148A) and GSK3β (K85A) display greater affinity to rat Arc protein than wildtype kinases. HA-tagged BIO- Arc protein was overexpressed in HEK293 cells together with a wildtype or KD form of FLAG-GSK3α (K148A) or a wildtype or KD form of FLAG-GSK3β (K85A) for 20 h. Cell lysates were then harvested and immediately subjected to avi-tag co-precipitation. Blots were probed with anti-FLAG antibody to detect overexpressed GSK3α/β proteins and probed with anti-HA antibody to detect recombinant Arc. (D,E) Rat and human Arc proteins are phosphorylated in vitro by recombinant GSK3α and GSK3β. The figures show representative results from the in vitro kinase assay. Arc proteins that were overexpressed in HEK293 cells were isolated and incubated with or without recombinant GSK3α or GSK3β. Arc phosphorylation was detected by autoradiography. The level of total Arc was estimated with Coomassie Brilliant Blue staining. The phosphorylated GSK3β band is visible beneath the Arc band (indicated by asterisk [*]).
    Streptavidin Coated Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GSK3α/β binds and phosphorylates Arc protein. (A) GSK3β directly associates with Arc protein. Purified GST-Arc protein of rat origin was incubated with protein lysate from rat hippocampus. GSK3α/β binding was determined by Western blot. (B) GSK3α/β may directly associate with Arc protein in cells. Rat and human Arc or β-Gal proteins that were overexpressed in HEK293 cells were captured on streptavidin-coated beads and incubated with mouse hippocampal lysate. Bait-kinase binding was estimated by probing the blot with anti-GSK3α/β antibody. Bait (e.g., biotinylated Arc or β-Gal) was detected using fluorescently labeled streptavidin. (C) Kinase dead (KD) mutants of GSK3α (K148A) and GSK3β (K85A) display greater affinity to rat Arc protein than wildtype kinases. HA-tagged BIO- Arc protein was overexpressed in HEK293 cells together with a wildtype or KD form of FLAG-GSK3α (K148A) or a wildtype or KD form of FLAG-GSK3β (K85A) for 20 h. Cell lysates were then harvested and immediately subjected to avi-tag co-precipitation. Blots were probed with anti-FLAG antibody to detect overexpressed GSK3α/β proteins and probed with anti-HA antibody to detect recombinant Arc. (D,E) Rat and human Arc proteins are phosphorylated in vitro by recombinant GSK3α and GSK3β. The figures show representative results from the in vitro kinase assay. Arc proteins that were overexpressed in HEK293 cells were isolated and incubated with or without recombinant GSK3α or GSK3β. Arc phosphorylation was detected by autoradiography. The level of total Arc was estimated with Coomassie Brilliant Blue staining. The phosphorylated GSK3β band is visible beneath the Arc band (indicated by asterisk [*]).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: GSK3α and GSK3β Phosphorylate Arc and Regulate its Degradation

    doi: 10.3389/fnmol.2017.00192

    Figure Lengend Snippet: GSK3α/β binds and phosphorylates Arc protein. (A) GSK3β directly associates with Arc protein. Purified GST-Arc protein of rat origin was incubated with protein lysate from rat hippocampus. GSK3α/β binding was determined by Western blot. (B) GSK3α/β may directly associate with Arc protein in cells. Rat and human Arc or β-Gal proteins that were overexpressed in HEK293 cells were captured on streptavidin-coated beads and incubated with mouse hippocampal lysate. Bait-kinase binding was estimated by probing the blot with anti-GSK3α/β antibody. Bait (e.g., biotinylated Arc or β-Gal) was detected using fluorescently labeled streptavidin. (C) Kinase dead (KD) mutants of GSK3α (K148A) and GSK3β (K85A) display greater affinity to rat Arc protein than wildtype kinases. HA-tagged BIO- Arc protein was overexpressed in HEK293 cells together with a wildtype or KD form of FLAG-GSK3α (K148A) or a wildtype or KD form of FLAG-GSK3β (K85A) for 20 h. Cell lysates were then harvested and immediately subjected to avi-tag co-precipitation. Blots were probed with anti-FLAG antibody to detect overexpressed GSK3α/β proteins and probed with anti-HA antibody to detect recombinant Arc. (D,E) Rat and human Arc proteins are phosphorylated in vitro by recombinant GSK3α and GSK3β. The figures show representative results from the in vitro kinase assay. Arc proteins that were overexpressed in HEK293 cells were isolated and incubated with or without recombinant GSK3α or GSK3β. Arc phosphorylation was detected by autoradiography. The level of total Arc was estimated with Coomassie Brilliant Blue staining. The phosphorylated GSK3β band is visible beneath the Arc band (indicated by asterisk [*]).

    Article Snippet: The supernatant was collected, loaded on streptavidin-coated beads (Dynabeads M280, Thermo Fisher Scientific), and rotated for 3 h at 4°C, followed by extensive washes in cell lysis buffer with 500 mM KCl and 2% Triton X-100.

    Techniques: Purification, Incubation, Binding Assay, Western Blot, Labeling, Hemagglutination Assay, Recombinant, In Vitro, Kinase Assay, Isolation, Autoradiography, Staining

    Non- N -glycosylated mTRAIL-R triggers ligand-dependent apoptosis in tunicamycin-treated cells. a MEFs were treated for 6 h with control or TU followed by stimulation with mTRAIL-SK (20 ng/ml) for the indicated times. Cell lysates were then immunoblotted as indicated. Caspase-8 and c-FLIP cleavage products are indicated by arrowheads. Asterisk indicates non-specific signal. Representative images of three independent experiments. b MEFs were exposed to TU or control for 7 h followed by Biotin-ILZ-hTRAIL (biot-ILZ-hTRAIL) (500 ng/ml) or CTRL treatment for 15 min. Cell lysates were subjected to streptavidin (Strep) pulldown and immunoblotted for mTRAIL-R and FADD under non-reducing conditions. Left panels: Strep pulldown. Right panels: Input. HMW high molecular weight. Representative images of two independent experiments. c MEFs were exposed to TU or control for 7 h followed by mTRAIL-SK (20 ng/ml) or CTRL treatment for 2 h. Cell lysates were subjected to FADD immunoprecipitation (IP:FADD) and immunoblotted as indicated. mTRAIL-R was detected under non-reducing conditions. Left panels: IP:FADD. Right panels: Input. Caspase-8 cleavage products are indicated by arrowheads. HMW high molecular weight. Asterisk indicates non-specific signal. Representative images of two independent experiments

    Journal: Cell Death & Disease

    Article Title: N-glycosylation of mouse TRAIL-R restrains TRAIL-induced apoptosis

    doi: 10.1038/s41419-018-0544-7

    Figure Lengend Snippet: Non- N -glycosylated mTRAIL-R triggers ligand-dependent apoptosis in tunicamycin-treated cells. a MEFs were treated for 6 h with control or TU followed by stimulation with mTRAIL-SK (20 ng/ml) for the indicated times. Cell lysates were then immunoblotted as indicated. Caspase-8 and c-FLIP cleavage products are indicated by arrowheads. Asterisk indicates non-specific signal. Representative images of three independent experiments. b MEFs were exposed to TU or control for 7 h followed by Biotin-ILZ-hTRAIL (biot-ILZ-hTRAIL) (500 ng/ml) or CTRL treatment for 15 min. Cell lysates were subjected to streptavidin (Strep) pulldown and immunoblotted for mTRAIL-R and FADD under non-reducing conditions. Left panels: Strep pulldown. Right panels: Input. HMW high molecular weight. Representative images of two independent experiments. c MEFs were exposed to TU or control for 7 h followed by mTRAIL-SK (20 ng/ml) or CTRL treatment for 2 h. Cell lysates were subjected to FADD immunoprecipitation (IP:FADD) and immunoblotted as indicated. mTRAIL-R was detected under non-reducing conditions. Left panels: IP:FADD. Right panels: Input. Caspase-8 cleavage products are indicated by arrowheads. HMW high molecular weight. Asterisk indicates non-specific signal. Representative images of two independent experiments

    Article Snippet: Biot-ILZ hTRAIL-bound complexes were precipitated using streptavidin magnetic beads (DynabeadsTM M-280 Streptavidin, Thermo Fisher Scientific, Waltham, MA USA) overnight at 4 °C.

    Techniques: Molecular Weight, Immunoprecipitation

    Proteomic screen identifies ESCRT components as EphB2 interactors. (A) Strategy of purification and identification of the interactome of biotinylated EphB2 by mass spectrometry. (B) Representative images showing clustering of biotinylated FLAG-Avi-EphB2-YFP fusion protein around streptavidin-conjugated Dynabeads (within 5 min, right, stippled line). This effect required EphB2 biotinylation (left). (C) Western blot analysis showing tyrosine autophosphorylation (detected by anti-phospho EphB2 [Y594] or 4G10 antibodies) of biotinylated (BirA + ) but not unbiotinylated (BirA – ) FLAG-Avi-EphB2 in response to incubation with streptavidin-conjugated Dynabeads. Unfused Fc protein was used as negative control. Similar results were observed in three independent replicates. (D) Bar graph showing the SILAC ratios of representative members of different groups of the top 30 enriched full-length EphB2 interactors ( n = 3, mean ± SEM). Yellow, biotinylated versus unbiotinylated full-length EphB2 (FL EphB2); blue, biotinylated EphB2-ΔC versus unbiotinylated full-length EphB2 (EphB2-ΔC); gray, biotinylated full-length EphB2 versus biotinylated EphB2-ΔC (EphB2-cyto). (E) Full list of ESCRT complex components identified in the proteomic screen as interactors of full-length EphB2 in at least one of the three replicates. (F) Validation of the interaction between EphB2 and endogenous STAM1 or VPS4A by co-IP/Western blot (WB) analysis in HEK293 cells stably expressing EphB2. (G) Validation of the interaction between overexpressed EphB2 and STAM1 in HeLa cells. (H) Representative images showing that endogenous STAM and CHMP4B colocalize with surface EphB2 in HeLa cells. STAM and CHMP4B levels at the plasma membrane were increased 3.5- and 2-fold, respectively (highlighted by red triangles), in EphB2 + cells (indicated by yellow stippled line in the merge) compared with untransfected cells (white stippled line). Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance

    doi: 10.1083/jcb.201601085

    Figure Lengend Snippet: Proteomic screen identifies ESCRT components as EphB2 interactors. (A) Strategy of purification and identification of the interactome of biotinylated EphB2 by mass spectrometry. (B) Representative images showing clustering of biotinylated FLAG-Avi-EphB2-YFP fusion protein around streptavidin-conjugated Dynabeads (within 5 min, right, stippled line). This effect required EphB2 biotinylation (left). (C) Western blot analysis showing tyrosine autophosphorylation (detected by anti-phospho EphB2 [Y594] or 4G10 antibodies) of biotinylated (BirA + ) but not unbiotinylated (BirA – ) FLAG-Avi-EphB2 in response to incubation with streptavidin-conjugated Dynabeads. Unfused Fc protein was used as negative control. Similar results were observed in three independent replicates. (D) Bar graph showing the SILAC ratios of representative members of different groups of the top 30 enriched full-length EphB2 interactors ( n = 3, mean ± SEM). Yellow, biotinylated versus unbiotinylated full-length EphB2 (FL EphB2); blue, biotinylated EphB2-ΔC versus unbiotinylated full-length EphB2 (EphB2-ΔC); gray, biotinylated full-length EphB2 versus biotinylated EphB2-ΔC (EphB2-cyto). (E) Full list of ESCRT complex components identified in the proteomic screen as interactors of full-length EphB2 in at least one of the three replicates. (F) Validation of the interaction between EphB2 and endogenous STAM1 or VPS4A by co-IP/Western blot (WB) analysis in HEK293 cells stably expressing EphB2. (G) Validation of the interaction between overexpressed EphB2 and STAM1 in HeLa cells. (H) Representative images showing that endogenous STAM and CHMP4B colocalize with surface EphB2 in HeLa cells. STAM and CHMP4B levels at the plasma membrane were increased 3.5- and 2-fold, respectively (highlighted by red triangles), in EphB2 + cells (indicated by yellow stippled line in the merge) compared with untransfected cells (white stippled line). Bars, 10 µm.

    Article Snippet: 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Techniques: Purification, Mass Spectrometry, Western Blot, Incubation, Flow Cytometry, Negative Control, Co-Immunoprecipitation Assay, Stable Transfection, Expressing

    Specificity analysis of ERaptD4 using flow cytometry, fluorescent microscopy and cytochemistry. ( a1-a2 ) Treatment of MCF-7 cells with fluorescent-tagged ERaptD4 produced a shift of 84% relative to unstained MCF-7 cells. Figure in inset provides the fluorescent microscopy image of the FAM-ERaptD4 treated cells. ( b1-b2 ) A shift of 20% is observed in MDA-MB-231 cell upon treatment with fluorescent-labelled ERaptD4. ( c1-c2 ) The treatment of MCF-7 cells with a random sequence (FAM-labelled non-enriched aptamer library) produced no shift, indicating the lack of binding by the random sequence. FAM-labelling of the non-enriched aptamer library is carried out using PCR amplification with FAM-forward primer and biotin-reverse primer. Strand separation is performed on streptavidin magnetic beads. ( d-e ) Aptacytochemistry of ERα-positive MCF-7 and ERα-deficient MDA-MB-231 cells. Size-bar on images a-c is 100 pixels (imaging at 63X). Size-bar on images d-e is 200 pixels (imaging at 20X).

    Journal: PLoS ONE

    Article Title: Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer

    doi: 10.1371/journal.pone.0153001

    Figure Lengend Snippet: Specificity analysis of ERaptD4 using flow cytometry, fluorescent microscopy and cytochemistry. ( a1-a2 ) Treatment of MCF-7 cells with fluorescent-tagged ERaptD4 produced a shift of 84% relative to unstained MCF-7 cells. Figure in inset provides the fluorescent microscopy image of the FAM-ERaptD4 treated cells. ( b1-b2 ) A shift of 20% is observed in MDA-MB-231 cell upon treatment with fluorescent-labelled ERaptD4. ( c1-c2 ) The treatment of MCF-7 cells with a random sequence (FAM-labelled non-enriched aptamer library) produced no shift, indicating the lack of binding by the random sequence. FAM-labelling of the non-enriched aptamer library is carried out using PCR amplification with FAM-forward primer and biotin-reverse primer. Strand separation is performed on streptavidin magnetic beads. ( d-e ) Aptacytochemistry of ERα-positive MCF-7 and ERα-deficient MDA-MB-231 cells. Size-bar on images a-c is 100 pixels (imaging at 63X). Size-bar on images d-e is 200 pixels (imaging at 20X).

    Article Snippet: The collected pools of enriched sequences from various rounds of SELEX screening (round 2, 4, 6, 8 and 9) were biotinylated (via PCR with biotinylated forward primer) and strand separated on magnetic streptavidin beads (Dynabeads® M-280 Streptavidin; Thermo Fisher Scientific) to obtain single stranded sense sequences.

    Techniques: Flow Cytometry, Cytometry, Microscopy, Produced, Multiple Displacement Amplification, Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification, Magnetic Beads, Imaging