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    Thermo Fisher dynabeads m 280 streptavidin
    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 M 280 Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 6896 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:

    (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.

    Journal: Journal of Extracellular Vesicles

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA

    doi: 10.3402/jev.v5.29828

    Figure Lengend Snippet: (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.

    Article Snippet: The CTB, AV or ST reaction mix was added to 30 µL equivalent of Dynabeads M280 Streptavidin (Thermo Fisher Scientific, Waltham, MA) that were pre-washed as per manufacturer's instruction.

    Techniques: Western Blot, CtB Assay, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Staining

    (a) SDS-PAGE analysis of MSC EVs extracted with membrane lipid-binding ligands, CTB, AV and ST, respectively. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilized with a magnet and the supernatant was collected as the “unbound” fraction. The beads were then washed twice and the wash solutions were collected as “wash 1” and “wash 2,” respectively. The beads were re-suspended in PBS as the “bound” fraction. The equivalent of 20% of the starting samples (input) and each of their respective “unbound,” “wash 1,” “wash 2” and “bound” fractions were resolved onto polyacrylamide gels and the gels were stained with silver. (b) Size distribution of MSC EVs by NanoSight. MSC EVs were diluted 1,000× with 0.22 µm filtered PBS. The size distribution of exosome was then measured using NanoSight LM10 and analysed by Nanoparticles Tracking Analysis software according to the manufacturer's protocol. (c) SEM analysis of MSC EVs that were extracted with CTB, AV and ST. MSC EV preparation was incubated with biotinylated CTB, AV, ST or without ligand and then streptavidin-coated polystyrene particles. The beads were then washed twice with PBS and resuspended in PBS before being spotted and left to dry onto carbon tape on aluminium stubs at 40°C. The stubs were sputter coated with 2 ηm of gold coating (Leica Biosystems) and imaged in a Jeol 6701FESEM. Scale bar=100 ηm.

    Journal: Journal of Extracellular Vesicles

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA

    doi: 10.3402/jev.v5.29828

    Figure Lengend Snippet: (a) SDS-PAGE analysis of MSC EVs extracted with membrane lipid-binding ligands, CTB, AV and ST, respectively. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilized with a magnet and the supernatant was collected as the “unbound” fraction. The beads were then washed twice and the wash solutions were collected as “wash 1” and “wash 2,” respectively. The beads were re-suspended in PBS as the “bound” fraction. The equivalent of 20% of the starting samples (input) and each of their respective “unbound,” “wash 1,” “wash 2” and “bound” fractions were resolved onto polyacrylamide gels and the gels were stained with silver. (b) Size distribution of MSC EVs by NanoSight. MSC EVs were diluted 1,000× with 0.22 µm filtered PBS. The size distribution of exosome was then measured using NanoSight LM10 and analysed by Nanoparticles Tracking Analysis software according to the manufacturer's protocol. (c) SEM analysis of MSC EVs that were extracted with CTB, AV and ST. MSC EV preparation was incubated with biotinylated CTB, AV, ST or without ligand and then streptavidin-coated polystyrene particles. The beads were then washed twice with PBS and resuspended in PBS before being spotted and left to dry onto carbon tape on aluminium stubs at 40°C. The stubs were sputter coated with 2 ηm of gold coating (Leica Biosystems) and imaged in a Jeol 6701FESEM. Scale bar=100 ηm.

    Article Snippet: The CTB, AV or ST reaction mix was added to 30 µL equivalent of Dynabeads M280 Streptavidin (Thermo Fisher Scientific, Waltham, MA) that were pre-washed as per manufacturer's instruction.

    Techniques: SDS Page, Binding Assay, CtB Assay, Incubation, Staining, Software

    Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.

    Journal: Journal of Virology

    Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction

    doi: 10.1128/JVI.01613-18

    Figure Lengend Snippet: Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.

    Article Snippet: For precipitation of the BD fusion proteins, streptavidin-coated magnetic beads (Dynabeads M-280-Streptavidin; Invitrogen, CA, USA) were used according to the manufacturer’s instructions.

    Techniques: Transfection, Western Blot, Labeling

    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

    Recruitment of the transcription machinery into the PIC. (A) Complexes were assembled on M280-streptavidin Dynabeads carrying a biotinylated DNA fragment from the plasmid pA4xMLΔ53, which contained four copies of the HNF-4 cognate site A and the adenovirus major late core promoter. After PIC formation, beads were washed and bound complexes were analyzed for their factor content by immunoblotting. PICs were formed with GTFs only (TBP, TFIIB, TFIIE, TFIIF, TFIIH, Pol II (lane 3); with GTFs and HNF-4 (lane 4); or with GTFs, HNF-4, and PC4 (lanes 2 and 5). Lane 2, control in which the indicated factors were incubated with beads only (no template DNA); lane 1, GTFs only (input). (B) Immobilized-template recruitment assays are as for panel A, except that all reaction mixtures contained TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Pol II, and TRAP/SMCC/Mediator, as shown in input lane 1. HNF-4 (lanes 3 and 5) and PC4 (lanes 4 and 5) were additionally added as indicated. (C) Immobilized-template recruitment assays are as for panel B, except that reaction mixtures in lanes 6 and 7 did not contain TFIID. HNF-4 (lanes 3, 5, and 7) and PC4 (lanes 4 to 7) were additionally added as indicated.

    Journal: Molecular and Cellular Biology

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4

    doi: 10.1128/MCB.22.15.5626-5637.2002

    Figure Lengend Snippet: Recruitment of the transcription machinery into the PIC. (A) Complexes were assembled on M280-streptavidin Dynabeads carrying a biotinylated DNA fragment from the plasmid pA4xMLΔ53, which contained four copies of the HNF-4 cognate site A and the adenovirus major late core promoter. After PIC formation, beads were washed and bound complexes were analyzed for their factor content by immunoblotting. PICs were formed with GTFs only (TBP, TFIIB, TFIIE, TFIIF, TFIIH, Pol II (lane 3); with GTFs and HNF-4 (lane 4); or with GTFs, HNF-4, and PC4 (lanes 2 and 5). Lane 2, control in which the indicated factors were incubated with beads only (no template DNA); lane 1, GTFs only (input). (B) Immobilized-template recruitment assays are as for panel A, except that all reaction mixtures contained TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Pol II, and TRAP/SMCC/Mediator, as shown in input lane 1. HNF-4 (lanes 3 and 5) and PC4 (lanes 4 and 5) were additionally added as indicated. (C) Immobilized-template recruitment assays are as for panel B, except that reaction mixtures in lanes 6 and 7 did not contain TFIID. HNF-4 (lanes 3, 5, and 7) and PC4 (lanes 4 to 7) were additionally added as indicated.

    Article Snippet: 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer.

    Techniques: Plasmid Preparation, Incubation

    HDGFRP2 associates with RBBP8/CtIP, heterochromatin-binding proteins and histone marks of transcriptionally silent chromatin. ( A ) Lysates of U2OS cells left untreated or treated with 1 μM camptothecin (CPT) for 1 h were immunoprecipitated with HDGFRP2 antibody (HDGFRP2) or IgG control and analysed by immunoblotting of indicated proteins. Similar results were obtained in a minimum of three independent experiments. ( B – D ) Co-localization of HDGFRP2 and RBBP8/CtiP was analysed using rabbit anti-HDGFRP2 and murine anti-RBBP8/CtiP antibodies and Duolink-based in situ proximity ligation assay (PLA) with appropriate secondary antibodies. U2OS cells were first transfected with control (C) or HDGFRP2 ( H4 ) siRNA for 72 h and then left untreated or subjected to treatment with 1 μM camptothecin (CPT) for 1 h with or without a 4 h recovery. Representative confocal images of PLA-stained cells, in which co-localization of the antibodies used results in red (624 nm) fluorescence are shown in (B). Median values of nuclear PLA signals from a minimum of 75 (control siRNA) cells per condition presented as floating-bar plots are shown in (C) and immunoblot demonstrating the efficacy of H4 siRNA in (D). The specificity of the PLA signal was confirmed by the significant reduction of the signal by H4 siRNA. Note, that the remaining HDGFR2 protein in H4 siRNA-treated cells can also result in the PLA signal. DNA was stained with DAPI to visualize the nuclei. ( E ) Biotin-conjugated histone peptides were incubated with U2OS lysates, followed by a pulldown with streptavidin-coated magnetic beads. Representative immunoblot of indicated proteins. The vertical line indicates where the blot was cut to remove non-essential bands after staining and exposure. ( F ) Co-localization of HDGFRP2 and H3K9me3 was analysed using goat anti-HDGFRP2 and rabbit anti-H3K9me3 antibodies and Duolink-based in situ PLA with appropriate secondary antibodies in U2OS cells left untreated or treated with 1 μM camptothecin (CPT) for 1 h with or without 1 h recovery. Median values of nuclear PLA signals from a minimum of 75 cells per condition (right) are shown as floating-bar plots. DNA was stained with DAPI to visualize the nuclei. Representative confocal images are shown in Supplementary Figure S2. Scale bars, 10 μm. * P

    Journal: Nucleic Acids Research

    Article Title: Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    doi: 10.1093/nar/gkv1526

    Figure Lengend Snippet: HDGFRP2 associates with RBBP8/CtIP, heterochromatin-binding proteins and histone marks of transcriptionally silent chromatin. ( A ) Lysates of U2OS cells left untreated or treated with 1 μM camptothecin (CPT) for 1 h were immunoprecipitated with HDGFRP2 antibody (HDGFRP2) or IgG control and analysed by immunoblotting of indicated proteins. Similar results were obtained in a minimum of three independent experiments. ( B – D ) Co-localization of HDGFRP2 and RBBP8/CtiP was analysed using rabbit anti-HDGFRP2 and murine anti-RBBP8/CtiP antibodies and Duolink-based in situ proximity ligation assay (PLA) with appropriate secondary antibodies. U2OS cells were first transfected with control (C) or HDGFRP2 ( H4 ) siRNA for 72 h and then left untreated or subjected to treatment with 1 μM camptothecin (CPT) for 1 h with or without a 4 h recovery. Representative confocal images of PLA-stained cells, in which co-localization of the antibodies used results in red (624 nm) fluorescence are shown in (B). Median values of nuclear PLA signals from a minimum of 75 (control siRNA) cells per condition presented as floating-bar plots are shown in (C) and immunoblot demonstrating the efficacy of H4 siRNA in (D). The specificity of the PLA signal was confirmed by the significant reduction of the signal by H4 siRNA. Note, that the remaining HDGFR2 protein in H4 siRNA-treated cells can also result in the PLA signal. DNA was stained with DAPI to visualize the nuclei. ( E ) Biotin-conjugated histone peptides were incubated with U2OS lysates, followed by a pulldown with streptavidin-coated magnetic beads. Representative immunoblot of indicated proteins. The vertical line indicates where the blot was cut to remove non-essential bands after staining and exposure. ( F ) Co-localization of HDGFRP2 and H3K9me3 was analysed using goat anti-HDGFRP2 and rabbit anti-H3K9me3 antibodies and Duolink-based in situ PLA with appropriate secondary antibodies in U2OS cells left untreated or treated with 1 μM camptothecin (CPT) for 1 h with or without 1 h recovery. Median values of nuclear PLA signals from a minimum of 75 cells per condition (right) are shown as floating-bar plots. DNA was stained with DAPI to visualize the nuclei. Representative confocal images are shown in Supplementary Figure S2. Scale bars, 10 μm. * P

    Article Snippet: Histone peptide pulldown Biotin-conjugated histone 3 (H3) peptides (Eurogentech) were immobilized on streptavidin magnetic beads (Life Technologies, Dynabeads M-280 Streptavidin) and incubated with nuclear extracts prepared as in the immunoprecipitation procedure but resolved in binding buffer (150 mM Hepes-KOH (pH7.6), 200 mM KCl, 1 mM MgCl2 , 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 1 mM DTT + Protease Inhibitors) for 2 h at 4°C.

    Techniques: Binding Assay, Cycling Probe Technology, Immunoprecipitation, In Situ, Proximity Ligation Assay, Transfection, Staining, Fluorescence, Incubation, Magnetic Beads

    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

    Purification of nuclear proteins binding to the SRE1-EBS-GRE elements of the human apoD promoter. (A) Nuclear extracts from normal growth (10% serum: +) or growth arrest (0.5% serum: −) conditions were incubated with either the biotinylated oligonucleotide bound to streptavidin beads (NE) or streptavidin beads alone (CTRL). The bound proteins were eluted and analyzed by SDS-PAGE and silver staining. The numbers and arrows on the gel indicate the excised bands. (B) Mass-spectrometry analysis of the eluted bands. Nuclear factors were identified by mass spectrometry (LC-MSMS) after tryptic digestion. STD: Standard molecular weight in kDa; this experiment was done in duplicate.

    Journal: Biochimica et Biophysica Acta. Molecular Cell Research

    Article Title: Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: Implication of PARP-1, APEX-1 and ERK1/2 catalytic activities

    doi: 10.1016/j.bbamcr.2010.04.011

    Figure Lengend Snippet: Purification of nuclear proteins binding to the SRE1-EBS-GRE elements of the human apoD promoter. (A) Nuclear extracts from normal growth (10% serum: +) or growth arrest (0.5% serum: −) conditions were incubated with either the biotinylated oligonucleotide bound to streptavidin beads (NE) or streptavidin beads alone (CTRL). The bound proteins were eluted and analyzed by SDS-PAGE and silver staining. The numbers and arrows on the gel indicate the excised bands. (B) Mass-spectrometry analysis of the eluted bands. Nuclear factors were identified by mass spectrometry (LC-MSMS) after tryptic digestion. STD: Standard molecular weight in kDa; this experiment was done in duplicate.

    Article Snippet: Streptavidin beads (500 μg; Dynabeads M-280 Strepavidin, Dynal Biotech, Oslo, Norway) were resuspended and incubated with 1500 pmoles of biotinylated oligonucleotides for 15 min in the buffer W & B (1 × ) (10 mM of Tris–HCl, pH 7.5, 1 M of NaCl and 1 mM of EDTA) as recommended by the manufacturer.

    Techniques: Purification, Binding Assay, Incubation, SDS Page, Silver Staining, Mass Spectrometry, Molecular Weight

    DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

    Journal: Nucleic Acids Research

    Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription

    doi: 10.1093/nar/gkw1208

    Figure Lengend Snippet: DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

    Article Snippet: The biotinylated ends were captured with 50 μl of streptavidin-coated paramagnetic beads (Dynabeads® M-280 Streptavidin, Invitrogen), and the DNA was then ligated to 60 pmol linker 2 in a 100 μl reaction volume containing 400 U of T4 DNA ligase by incubation at 16°C for 4 h. The precipitate was gently resuspended in 100 μl of 0.15 M NaOH at room temperature for 10 min and eluted with 30 μl 10 mM Tris–Cl (pH 8.0).

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, In Situ, Electrophoresis, Agarose Gel Electrophoresis, Fluorescence, Labeling, Ligation, Cell Culture

    PKR binding to modified siRNAs containing 8-alkoxyadenosine switches. (A) Biotinylated siRNAs were bound to magnetic streptavidin beads and treated with lysates from U87 cells treated with IFN-α. The amount of PKR retained was determined by western

    Journal: Journal of the American Chemical Society

    Article Title: Promiscuous 8-Alkoxyadenosines in the Guide Strand of an SiRNA: Modulation of Silencing Efficacy and Off-Pathway Protein Binding

    doi: 10.1021/ja307102g

    Figure Lengend Snippet: PKR binding to modified siRNAs containing 8-alkoxyadenosine switches. (A) Biotinylated siRNAs were bound to magnetic streptavidin beads and treated with lysates from U87 cells treated with IFN-α. The amount of PKR retained was determined by western

    Article Snippet: Magnetic streptavidin beads (0.5 mg, 50 μL) (Dynabeads M-280, Invitrogen) were prepared for RNA manipulation according to the manufacturer’s protocol.

    Techniques: Binding Assay, Modification, Western Blot

    Discovery of allele-specific binding proteins at cis -regulatory variants. ( A ) Workflow: (1) cis-regulatory variant prediction at disease associated variants ( PPARG ) in high LD ( r 2 ≥ 0.7 ( 6 )) by integrating bioinformatics phylogenetic TFBS module complexity analysis and regulatory chromatin marks; (2) protein–DNA binding assessed by Cy5 labeled oligonucleotides matching the risk and nonrisk allele, respectively, in electrophoretic mobility shift assay (EMSA); (3) protein enrichment with biotin (bio) labeled oligonucleotides on streptavidin-beads (str) and elution of native protein complexes with increasing concentration of NaCl; (4) protein–DNA binding in eluted fractions; (5) protein identification and quantification by LC–MS/MS and subsequent label-free quantitative analysis; and (6) molecular mechanisms, experimental and genetics verification of significant allele-specific binding transcription factors and related coregulators. (B–D) Bioinformatics and public domain epigenomic marks of regulatory regions infer the cis -regulatory variant rs7647481 at the PPARG locus (related to Supplementary Figure S1 ). ( B ) PMCA analysis of cross-species TFBS pattern conservation predicted six indicated candidate cis -regulatory SNPs at complex regions ( 6 ) (red) out of 23 noncoding proxy SNPs ( r 2 ≥ 0.7 ( 6 )) at the type 2 diabetes (T2D) associated PPARG locus (tagSNP rs1801282). ( C ) Overlap of six variants identified in (B) with H3K27ac (histone H3-lysine 27 acetylation), H3K4me1 and H3K4me2 (histone H3–lysine 4 mono- and di-methylation) histone modification regions at the PPARG locus during adipogenic differentiation of primary human adipocyte stem cells ( 36 ), GSE21366, genomic coordinates are given conform to hg19. ( D ) Localization of cis- regulatory (red) and non cis -regulatory (grey) variants subjected to workflow (A2–6) relative to transcriptional start site of the PPARG1–3 mRNA isoforms. rs7647481 overlapping with both, day 3 and day 9, tested late stage of adipogenesis histone modification regions (Figure 1C ) and with adipocyte DNase-seq regions (see Supplementary Figure S1 ). * rs4684847 previously identified as specifically overlapping with homeobox TFBS ( 6 ). Blue boxes = coding exons, dashed white boxes = untranslated exons, blue lines = introns, black arrows = promoters.

    Journal: Nucleic Acids Research

    Article Title: Allele-specific quantitative proteomics unravels molecular mechanisms modulated by cis-regulatory PPARG locus variation

    doi: 10.1093/nar/gkx105

    Figure Lengend Snippet: Discovery of allele-specific binding proteins at cis -regulatory variants. ( A ) Workflow: (1) cis-regulatory variant prediction at disease associated variants ( PPARG ) in high LD ( r 2 ≥ 0.7 ( 6 )) by integrating bioinformatics phylogenetic TFBS module complexity analysis and regulatory chromatin marks; (2) protein–DNA binding assessed by Cy5 labeled oligonucleotides matching the risk and nonrisk allele, respectively, in electrophoretic mobility shift assay (EMSA); (3) protein enrichment with biotin (bio) labeled oligonucleotides on streptavidin-beads (str) and elution of native protein complexes with increasing concentration of NaCl; (4) protein–DNA binding in eluted fractions; (5) protein identification and quantification by LC–MS/MS and subsequent label-free quantitative analysis; and (6) molecular mechanisms, experimental and genetics verification of significant allele-specific binding transcription factors and related coregulators. (B–D) Bioinformatics and public domain epigenomic marks of regulatory regions infer the cis -regulatory variant rs7647481 at the PPARG locus (related to Supplementary Figure S1 ). ( B ) PMCA analysis of cross-species TFBS pattern conservation predicted six indicated candidate cis -regulatory SNPs at complex regions ( 6 ) (red) out of 23 noncoding proxy SNPs ( r 2 ≥ 0.7 ( 6 )) at the type 2 diabetes (T2D) associated PPARG locus (tagSNP rs1801282). ( C ) Overlap of six variants identified in (B) with H3K27ac (histone H3-lysine 27 acetylation), H3K4me1 and H3K4me2 (histone H3–lysine 4 mono- and di-methylation) histone modification regions at the PPARG locus during adipogenic differentiation of primary human adipocyte stem cells ( 36 ), GSE21366, genomic coordinates are given conform to hg19. ( D ) Localization of cis- regulatory (red) and non cis -regulatory (grey) variants subjected to workflow (A2–6) relative to transcriptional start site of the PPARG1–3 mRNA isoforms. rs7647481 overlapping with both, day 3 and day 9, tested late stage of adipogenesis histone modification regions (Figure 1C ) and with adipocyte DNase-seq regions (see Supplementary Figure S1 ). * rs4684847 previously identified as specifically overlapping with homeobox TFBS ( 6 ). Blue boxes = coding exons, dashed white boxes = untranslated exons, blue lines = introns, black arrows = promoters.

    Article Snippet: According to the manufacturer΄s instructions, streptavidin coupled magnetic beads (Dynabeads M-280, Invitrogen, Darmstadt, Germany) were washed and collected using Bind & Wash buffer (Dynabeads M-280, Invitrogen, Darmstadt, Germany) and Magnetic particle separator (Magna-Sep™, Invitrogen, Darmstadt, Germany), discarding the supernatant.

    Techniques: Binding Assay, Variant Assay, Labeling, Electrophoretic Mobility Shift Assay, Protein Enrichment, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Methylation, Modification

    Outline of TRE5-A bsr retrotransposition profiling. Note that TRE5-A always integrates in an orientation-specific manner with the 5’ end of the retrotransposon facing the 5’ end of the targeted tRNA gene. LAM-PCR was performed on genomic DNA prepared from a pool of blasticidin-resistant clones. The 5'-biotinylated primer bound selectively to the codon-adapted ORF1 gene of the TRE5-A bsr element and did not recognize ORF1 genes of endogenous TRE5-A elements. The resulting linear, single-stranded LAM-PCR products were immobilized on streptavidin beads and washed extensively. To perform profiling of TRE5-A bsr insertions at tRNA genes, exponential PCRs were performed in parallel reactions with primers specific for selected tRNA gene families (“tDNA primer library”). To profile TRE5-A bsr insertions at any position in the genome, second-strand synthesis was initiated with a random hexamer primer linked to a unique adapter oligonucleotide (adapter-N 6 ). Next, exponential PCR was performed with the adapter primer and an ORF1-specific primer to yield an “adapter primer library”. SD, splice donor site; SA, splice acceptor site.

    Journal: PLoS ONE

    Article Title: TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element

    doi: 10.1371/journal.pone.0175729

    Figure Lengend Snippet: Outline of TRE5-A bsr retrotransposition profiling. Note that TRE5-A always integrates in an orientation-specific manner with the 5’ end of the retrotransposon facing the 5’ end of the targeted tRNA gene. LAM-PCR was performed on genomic DNA prepared from a pool of blasticidin-resistant clones. The 5'-biotinylated primer bound selectively to the codon-adapted ORF1 gene of the TRE5-A bsr element and did not recognize ORF1 genes of endogenous TRE5-A elements. The resulting linear, single-stranded LAM-PCR products were immobilized on streptavidin beads and washed extensively. To perform profiling of TRE5-A bsr insertions at tRNA genes, exponential PCRs were performed in parallel reactions with primers specific for selected tRNA gene families (“tDNA primer library”). To profile TRE5-A bsr insertions at any position in the genome, second-strand synthesis was initiated with a random hexamer primer linked to a unique adapter oligonucleotide (adapter-N 6 ). Next, exponential PCR was performed with the adapter primer and an ORF1-specific primer to yield an “adapter primer library”. SD, splice donor site; SA, splice acceptor site.

    Article Snippet: A typical reaction was conducted with 100 cycles of annealing at 58°C for 30 sec and elongation at 68°C for 45 sec. Biotinylated single-stranded PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany) and then immobilized onto streptavidin-conjugated magnetic beads (Dynabeads® M-280, Invitrogen) to remove contaminating genomic DNA.

    Techniques: Laser Capture Microdissection, Polymerase Chain Reaction, Clone Assay, Random Hexamer Labeling

    Histone fate after in vitro chromatin resection by the Sgs1-Dna2 pathway. A , native PAGE of a 500-bp DNA fragment harboring a central, 601-positioning sequence reconstituted into mononucleosomes by salt step dialysis at different ratios ( r ) of histone octamers to DNA. Note that the minor nucleosome species is likely to represent a nucleosome assembled on the DNA end. B and C , chromatin resection time course with 3′-radiolabeled chromatin and reactions that contain Mre11-Rad50-Xrs2, Sgs1, Top3-Rmi complex, Dna2, and RPA. C, addition of streptavidin-coated magnetic beads inhibit chromatin resection on one strand. Note the appearance of slower migrating ssDNA. D, analysis of DNA and protein content following magnetic DNA pulldown after chromatin resection. Left panel , radiolabel analysis of DNA before (−) and after (+) resection. One sample was also treated with EcoRI prior to resection and the released DNA was analyzed. Right panel , histone immunoblotting of bead bound ( B ) and unbound ( U ) fractions before and after chromatin resection.

    Journal: The Journal of Biological Chemistry

    Article Title: Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair *

    doi: 10.1074/jbc.M117.776369

    Figure Lengend Snippet: Histone fate after in vitro chromatin resection by the Sgs1-Dna2 pathway. A , native PAGE of a 500-bp DNA fragment harboring a central, 601-positioning sequence reconstituted into mononucleosomes by salt step dialysis at different ratios ( r ) of histone octamers to DNA. Note that the minor nucleosome species is likely to represent a nucleosome assembled on the DNA end. B and C , chromatin resection time course with 3′-radiolabeled chromatin and reactions that contain Mre11-Rad50-Xrs2, Sgs1, Top3-Rmi complex, Dna2, and RPA. C, addition of streptavidin-coated magnetic beads inhibit chromatin resection on one strand. Note the appearance of slower migrating ssDNA. D, analysis of DNA and protein content following magnetic DNA pulldown after chromatin resection. Left panel , radiolabel analysis of DNA before (−) and after (+) resection. One sample was also treated with EcoRI prior to resection and the released DNA was analyzed. Right panel , histone immunoblotting of bead bound ( B ) and unbound ( U ) fractions before and after chromatin resection.

    Article Snippet: Chromatin was magnetically precipitated with streptavidin-coated beads (Dynabeads M-280 Streptavidin, Invitrogen) and beads were then washed twice with 10 m m Tris-HCl, pH 7.5, 50 m m NaCl, 0.5 mg/ml of BSA to remove unbound chromatin.

    Techniques: In Vitro, Clear Native PAGE, Sequencing, Recombinase Polymerase Amplification, Magnetic Beads

    Biochemical characterization of reconstituted mononucleosomes. A, native PAGE of the indicated nucleic acid with increasing histone ratios ( r ) after reconstitution by salt step dialysis. Note that ssDNA stains less intensely with ethidium bromide. B , 4% native PAGE of ssDNA-histone octamer reconstitutions, using radiolabeled DNA fragments of varying length. r , histone octamer:DNA molar ratio. C, the stability of double-stranded and single-stranded nucleosomes after a 1-h incubation at the indicated range of temperatures, analyzed by separation on a 4% native PAGE. D, 200-nt ssDNA or 200-bp dsDNA chromatin reconstitutions were immobilized on streptavidin-coated magnetic beads, and histone content was analyzed after magnetic pulldown by SDS-PAGE. E , the indicated histone complexes were used in chromatin reconstitution reactions with 150-bp or 150-nt DNA fragments. Reconstitutions were analyzed by 4% native PAGE.

    Journal: The Journal of Biological Chemistry

    Article Title: Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair *

    doi: 10.1074/jbc.M117.776369

    Figure Lengend Snippet: Biochemical characterization of reconstituted mononucleosomes. A, native PAGE of the indicated nucleic acid with increasing histone ratios ( r ) after reconstitution by salt step dialysis. Note that ssDNA stains less intensely with ethidium bromide. B , 4% native PAGE of ssDNA-histone octamer reconstitutions, using radiolabeled DNA fragments of varying length. r , histone octamer:DNA molar ratio. C, the stability of double-stranded and single-stranded nucleosomes after a 1-h incubation at the indicated range of temperatures, analyzed by separation on a 4% native PAGE. D, 200-nt ssDNA or 200-bp dsDNA chromatin reconstitutions were immobilized on streptavidin-coated magnetic beads, and histone content was analyzed after magnetic pulldown by SDS-PAGE. E , the indicated histone complexes were used in chromatin reconstitution reactions with 150-bp or 150-nt DNA fragments. Reconstitutions were analyzed by 4% native PAGE.

    Article Snippet: Chromatin was magnetically precipitated with streptavidin-coated beads (Dynabeads M-280 Streptavidin, Invitrogen) and beads were then washed twice with 10 m m Tris-HCl, pH 7.5, 50 m m NaCl, 0.5 mg/ml of BSA to remove unbound chromatin.

    Techniques: Clear Native PAGE, Incubation, Magnetic Beads, SDS Page

    Components and performance of the INTACT system ( A ) Confocal projection of the differentiation zone of an ADF8p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in hair cells. GFP signal is shown in green and propidium iodide staining of cell walls is shown in red. ( B ) Confocal projection of the differentiation zone of an GL2p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in non-hair cells. ( C ) Confocal section of the post-meristematic region of a GL2p : NTF/ACT2p : BirA transgenic root. ( D ) Fluorescence micrograph of nuclei (one is shown in inset) isolated from ADF8p : NTF/ACT2p : BirA transgenic roots and incubated with streptavidin Dynabeads. GFP and beads are shown in green and DAPI staining of DNA is shown in blue. ( E ) Streptavidin western blot of whole cell extracts (input) and anti-GFP immunoprecipitates (IP) from roots of ACT2p : BirA, ADF8p : NTF/ACT2p : BirA , and GL2p : NTF/ACT2p : BirA plants. Top and bottom bands in each lane are endogenous biotinylated proteins and the middle band is the 42 kD NTF. ( F .

    Journal: Developmental cell

    Article Title: A simple method for gene expression and chromatin profiling of individual cell types within a tissue

    doi: 10.1016/j.devcel.2010.05.013

    Figure Lengend Snippet: Components and performance of the INTACT system ( A ) Confocal projection of the differentiation zone of an ADF8p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in hair cells. GFP signal is shown in green and propidium iodide staining of cell walls is shown in red. ( B ) Confocal projection of the differentiation zone of an GL2p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in non-hair cells. ( C ) Confocal section of the post-meristematic region of a GL2p : NTF/ACT2p : BirA transgenic root. ( D ) Fluorescence micrograph of nuclei (one is shown in inset) isolated from ADF8p : NTF/ACT2p : BirA transgenic roots and incubated with streptavidin Dynabeads. GFP and beads are shown in green and DAPI staining of DNA is shown in blue. ( E ) Streptavidin western blot of whole cell extracts (input) and anti-GFP immunoprecipitates (IP) from roots of ACT2p : BirA, ADF8p : NTF/ACT2p : BirA , and GL2p : NTF/ACT2p : BirA plants. Top and bottom bands in each lane are endogenous biotinylated proteins and the middle band is the 42 kD NTF. ( F .

    Article Snippet: Twenty-five microliters of Invitrogen M-280 streptavidin-coated dynabeads (~1.5 × 107 beads) were added to the nuclear suspensions and this mixture was rotated at 4° C for 30 min to allow binding of beads to the biotinylated nuclei.

    Techniques: Transgenic Assay, Expressing, Staining, Fluorescence, Isolation, Incubation, Western Blot