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    Thermo Fisher dynabeads m280 streptavidin coated beads thermo fisher
    Brusatol can directly target the PI3Kγ isoform. a  IC50 of Brusatol was determined in these NPC cell lines (C17, NPC43, and NPC53) and Brusatol-sensitive SU-DHL-4 cells at 72 h post-treatment.  b  These cell lines were harvested and detected the endogenous expressions of indicated proteins with western blot analysis.  c  The lysates of SU-DHL-4 cells were incubated with biotin-conjugated 51048 compound together in the absence or presence of Brusatol, then the immunoprecipitated complex was detected with western blot.  d  In vitro expressed and purified GST-tagged PI3Kγ protein was incubated with 0, 300 μM, or 500 μM biotin-conjugated 51052 compound as well as Dynabeads M-280 Streptavidin. Then the binding protein was examined with western blot analysis.  e  A schematic diagram shows the target site of  PIK3CG  genes using the CRISPR/Cas9 system.  f  A Surveyor mutation detection assay was performed to verify whether the  PIK3CG  gene was mutated in knock-out (KO) Raji cells. The control cell line (sgVec) by transfecting empty plasmids were used as control. The yellow arrows indicated the truncated fragments.  g  The indicated proteins were detected in knock-out Raji cells by western blot analysis.  h  Knock-out Raji cells were untreated or treated with 100 nM of Brusatol for 72 h, then cells were harvested and determined the expressions of PI3K/AKT associated proteins with western blot.
    Dynabeads M280 Streptavidin Coated Beads Thermo Fisher, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Brusatol can directly target the PI3Kγ isoform. a  IC50 of Brusatol was determined in these NPC cell lines (C17, NPC43, and NPC53) and Brusatol-sensitive SU-DHL-4 cells at 72 h post-treatment.  b  These cell lines were harvested and detected the endogenous expressions of indicated proteins with western blot analysis.  c  The lysates of SU-DHL-4 cells were incubated with biotin-conjugated 51048 compound together in the absence or presence of Brusatol, then the immunoprecipitated complex was detected with western blot.  d  In vitro expressed and purified GST-tagged PI3Kγ protein was incubated with 0, 300 μM, or 500 μM biotin-conjugated 51052 compound as well as Dynabeads M-280 Streptavidin. Then the binding protein was examined with western blot analysis.  e  A schematic diagram shows the target site of  PIK3CG  genes using the CRISPR/Cas9 system.  f  A Surveyor mutation detection assay was performed to verify whether the  PIK3CG  gene was mutated in knock-out (KO) Raji cells. The control cell line (sgVec) by transfecting empty plasmids were used as control. The yellow arrows indicated the truncated fragments.  g  The indicated proteins were detected in knock-out Raji cells by western blot analysis.  h  Knock-out Raji cells were untreated or treated with 100 nM of Brusatol for 72 h, then cells were harvested and determined the expressions of PI3K/AKT associated proteins with western blot.

    Journal: Communications Biology

    Article Title: Quassinoid analogs with enhanced efficacy for treatment of hematologic malignancies target the PI3Kγ isoform

    doi: 10.1038/s42003-020-0996-z

    Figure Lengend Snippet: Brusatol can directly target the PI3Kγ isoform. a IC50 of Brusatol was determined in these NPC cell lines (C17, NPC43, and NPC53) and Brusatol-sensitive SU-DHL-4 cells at 72 h post-treatment. b These cell lines were harvested and detected the endogenous expressions of indicated proteins with western blot analysis. c The lysates of SU-DHL-4 cells were incubated with biotin-conjugated 51048 compound together in the absence or presence of Brusatol, then the immunoprecipitated complex was detected with western blot. d In vitro expressed and purified GST-tagged PI3Kγ protein was incubated with 0, 300 μM, or 500 μM biotin-conjugated 51052 compound as well as Dynabeads M-280 Streptavidin. Then the binding protein was examined with western blot analysis. e A schematic diagram shows the target site of PIK3CG genes using the CRISPR/Cas9 system. f A Surveyor mutation detection assay was performed to verify whether the PIK3CG gene was mutated in knock-out (KO) Raji cells. The control cell line (sgVec) by transfecting empty plasmids were used as control. The yellow arrows indicated the truncated fragments. g The indicated proteins were detected in knock-out Raji cells by western blot analysis. h Knock-out Raji cells were untreated or treated with 100 nM of Brusatol for 72 h, then cells were harvested and determined the expressions of PI3K/AKT associated proteins with western blot.

    Article Snippet: Pull-down assayThirty microgram of purified GST-tagged proteins were pre-cleared with Dynabeads M-280 Streptavidin (Invitrogen, Carlsbad, CA, USA).

    Techniques: Western Blot, Incubation, Immunoprecipitation, In Vitro, Purification, Binding Assay, CRISPR, Mutagenesis, Detection Assay, Knock-Out

    Gwl promotes checkpoint recovery. (A) As in , biotinylated dA-dT oligos bound to M-280 streptavidin beads were added to extracts for 30 min to activate the DNA damage checkpoint and then removed with a magnet to allow recovery. As indicated,

    Journal: Cell Cycle

    Article Title: A novel role for Greatwall kinase in recovery from DNA damage

    doi: 10.4161/cc.9.21.13632

    Figure Lengend Snippet: Gwl promotes checkpoint recovery. (A) As in , biotinylated dA-dT oligos bound to M-280 streptavidin beads were added to extracts for 30 min to activate the DNA damage checkpoint and then removed with a magnet to allow recovery. As indicated,

    Article Snippet: For checkpoint activation and recovery, biotinylated dA-dT oligos were pre-bound to M-280 streptavidin Dynabeads (Invitrogen) following the standard protocol provided by the manufacturer, and the beads were then added to the extracts to produce a final concentration of 20 ug/ml dA-dT.

    Techniques:

    Pull-down assays for in vitro interactions of TIR1 WT and TIR1 F79A with the IAA7 DII peptide in the presence of IAA and 21 . Input is an aliquot of TIR1 WT -FLAG and TIR1 F79A -FLAG solution used for pull-down. The assays were performed with the biotinylated IAA7 DII peptide bound to Dynabeads M-280 streptavidin beads in the presence of IAA or 21 at the concentrations indicated on the top of each lane, and immunoblotting was conducted with an anti-FLAG antibody. In the lower graph, each bar represents the signal intensity of the immunoreactive band corresponding to the upper blot. Data are expressed as the mean � SE of three independent experiments.

    Journal: Plant and Cell Physiology

    Article Title: A Super Strong Engineered Auxin–TIR1 Pair

    doi: 10.1093/pcp/pcy127

    Figure Lengend Snippet: Pull-down assays for in vitro interactions of TIR1 WT and TIR1 F79A with the IAA7 DII peptide in the presence of IAA and 21 . Input is an aliquot of TIR1 WT -FLAG and TIR1 F79A -FLAG solution used for pull-down. The assays were performed with the biotinylated IAA7 DII peptide bound to Dynabeads M-280 streptavidin beads in the presence of IAA or 21 at the concentrations indicated on the top of each lane, and immunoblotting was conducted with an anti-FLAG antibody. In the lower graph, each bar represents the signal intensity of the immunoreactive band corresponding to the upper blot. Data are expressed as the mean � SE of three independent experiments.

    Article Snippet: Pull-down assays were performed using biotinyl-DII [biotinyl-(NH)-AKAQVVGWPPVRNYRKN] peptide, Dynabeads M-280 streptavidin beads (Invitrogen) and C-terminally FLAG-tagged TIR1 proteins which were synthesized with a wheat germ extract cell-free system (NUProtein) ( , ). mRNAs for FLAG-tagged TIR1WT and TIR1F79A proteins were synthesized by reverse transcription with PCR products amplified by first- and second-strand PCR according to the manufacturer’s instruction (NUProtein).

    Techniques: In Vitro

    Streptavidin and His-tag pull down assay of CFPS malE-en2NTS 1 and E. coli expressed en2NTS 1 . Binding assay, testing capability of binding to Biotin-NT using Streptavidin dynabeads in combination with ( A ). CFPS produced malE-en2NTS 1 and ( B ). The E. coli expressed en2NTS 1 control. Each graph reads 1. Receptor alone. 2. Biotin-NT plus receptor. 3. Biotin NT, receptor and unlabelled NT 8–13 competitor. Binding assay using His-tag isolation dynabeads in combination with ( C ). CFPS produced malE-en2NTS 1 ( D ). E. coli expressed en2NTS 1 . Each graph reads 1. Receptor alone. 2. A647-NT 8–13 plus receptor. 3. A647-NT 8–13 , receptor and NT 8–13 competitor. Data is presented as triplicate experiments ± SEM.

    Journal: Scientific Reports

    Article Title: Characterisation of a cell-free synthesised G-protein coupled receptor

    doi: 10.1038/s41598-017-01227-z

    Figure Lengend Snippet: Streptavidin and His-tag pull down assay of CFPS malE-en2NTS 1 and E. coli expressed en2NTS 1 . Binding assay, testing capability of binding to Biotin-NT using Streptavidin dynabeads in combination with ( A ). CFPS produced malE-en2NTS 1 and ( B ). The E. coli expressed en2NTS 1 control. Each graph reads 1. Receptor alone. 2. Biotin-NT plus receptor. 3. Biotin NT, receptor and unlabelled NT 8–13 competitor. Binding assay using His-tag isolation dynabeads in combination with ( C ). CFPS produced malE-en2NTS 1 ( D ). E. coli expressed en2NTS 1 . Each graph reads 1. Receptor alone. 2. A647-NT 8–13 plus receptor. 3. A647-NT 8–13 , receptor and NT 8–13 competitor. Data is presented as triplicate experiments ± SEM.

    Article Snippet: For malE-en2NTS1 pulldown experiments, two different dynabead matrices were used; Streptavidin coupled M-280 dynabeads or His-tag isolation and pulldown dynabeads (Life Technologies).

    Techniques: Pull Down Assay, Binding Assay, Produced, Isolation

    Effect of the concentration of sodium chloride, potassium chloride and magnesium chloride on binding of PNA I . Binding of biotin-labeled PNA I complementary to the inverted repeat at base pairs 1545–1562 within pUC19 and a control PNA that was not complementary to any sequence within pUC19 was carried out at 37°C for 30 min either before ( A ) or after ( B ) addition of the indicated monovalent or divalent salts. Complexes between plasmid and PNA were isolated by biotin–streptavidin affinity capture as described in Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: Strand invasion by mixed base PNAs and a PNA-peptide chimera

    doi:

    Figure Lengend Snippet: Effect of the concentration of sodium chloride, potassium chloride and magnesium chloride on binding of PNA I . Binding of biotin-labeled PNA I complementary to the inverted repeat at base pairs 1545–1562 within pUC19 and a control PNA that was not complementary to any sequence within pUC19 was carried out at 37°C for 30 min either before ( A ) or after ( B ) addition of the indicated monovalent or divalent salts. Complexes between plasmid and PNA were isolated by biotin–streptavidin affinity capture as described in Materials and Methods.

    Article Snippet: Affinity capture of plasmid DNA employed Dynabeads M-280 derivatized with streptavidin (Dynal, Oslo, Norway) as a matrix for separation of plasmids bound to biotin-labeled PNAs. pUC19 (40 nM) was mixed with biotin-labeled PNAs (500 nM) in 10 mM Tris–HCl, pH 8.0, at 37°C for 30 min prior to addition of streptavidin-coated beads.

    Techniques: Concentration Assay, Binding Assay, Labeling, Sequencing, Plasmid Preparation, Isolation