streptavidin coated magnetic beads  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin coated magnetic beads
    Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or <t>streptavidin-precoated</t> (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.
    Streptavidin Coated Magnetic Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3"

    Article Title: Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011941

    Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or streptavidin-precoated (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.
    Figure Legend Snippet: Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or streptavidin-precoated (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Derivative Assay, Negative Control, Purification, Injection

    Affinity matured anti-VEGF-C scFvs possess a higher affinity. (A, B) ELISA analysis of bacterial supernatant from randomly picked affinity matured clones after 1 to 3 rounds of selection on biotinylated (A) P. pastoris -derived or (B) mammalian cell-derived ΔNΔC-VEGF-C. (C) BIAcore profiles of monomeric affinity matured anti-VEGF-C scFvs. Monomeric fractions of protein-A purified scFv were prepared by FPLC and injected as 2-fold dilution series on a streptavidin-sensorchip coated with 2000 RU biotinylated ΔNΔC-VEGF-C derived from mammalian cells.
    Figure Legend Snippet: Affinity matured anti-VEGF-C scFvs possess a higher affinity. (A, B) ELISA analysis of bacterial supernatant from randomly picked affinity matured clones after 1 to 3 rounds of selection on biotinylated (A) P. pastoris -derived or (B) mammalian cell-derived ΔNΔC-VEGF-C. (C) BIAcore profiles of monomeric affinity matured anti-VEGF-C scFvs. Monomeric fractions of protein-A purified scFv were prepared by FPLC and injected as 2-fold dilution series on a streptavidin-sensorchip coated with 2000 RU biotinylated ΔNΔC-VEGF-C derived from mammalian cells.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Clone Assay, Selection, Derivative Assay, Purification, Fast Protein Liquid Chromatography, Injection

    2) Product Images from "Overexpression of TGF-?1 Gene Induces Cell Surface Localized Glucose-Regulated Protein 78-Associated Latency-Associated Peptide/TGF-?"

    Article Title: Overexpression of TGF-?1 Gene Induces Cell Surface Localized Glucose-Regulated Protein 78-Associated Latency-Associated Peptide/TGF-?

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0904121

    Identification of surface LAP/TGF-β–associated proteins. A , P3U1–TGF-β clone No. 16 cells or parent P3U1 cells were first surface labeled with biotin. The cell lysates were immunoprecipitated with anti-LAP mAb. The elutes further separated with SA magnetic beads, and blotted with SA-HRP. SA-bound fractions ( lanes 1 , 2 ) and -unbound fractions ( lanes 3 , 4 ) from P3U1 cells ( lanes 1 , 3 ) or P3U1–TGF-β cells ( lanes 2 , 4 ). B , Silver staining of the SA-bound fractions from P3U1 cells ( lane 1 ) or from P3U1–TGF-β cells ( lane 2 ). The band at the arrow was cut and subjected to LC/MS analysis. SA, streptavidin.
    Figure Legend Snippet: Identification of surface LAP/TGF-β–associated proteins. A , P3U1–TGF-β clone No. 16 cells or parent P3U1 cells were first surface labeled with biotin. The cell lysates were immunoprecipitated with anti-LAP mAb. The elutes further separated with SA magnetic beads, and blotted with SA-HRP. SA-bound fractions ( lanes 1 , 2 ) and -unbound fractions ( lanes 3 , 4 ) from P3U1 cells ( lanes 1 , 3 ) or P3U1–TGF-β cells ( lanes 2 , 4 ). B , Silver staining of the SA-bound fractions from P3U1 cells ( lane 1 ) or from P3U1–TGF-β cells ( lane 2 ). The band at the arrow was cut and subjected to LC/MS analysis. SA, streptavidin.

    Techniques Used: Labeling, Immunoprecipitation, Magnetic Beads, Silver Staining, Liquid Chromatography with Mass Spectroscopy

    3) Product Images from "The FANCM/FAAP24 Complex is Required for the DNA Inter-strand Crosslink-Induced Checkpoint Response"

    Article Title: The FANCM/FAAP24 Complex is Required for the DNA Inter-strand Crosslink-Induced Checkpoint Response

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2010.07.005

    RPA binding to ICL DNA is dependent on FAAP24 (A) Migration of ICL DNA on a denaturing gel. (B) Immunoblot showing that FAAP24 wild-type (WT) but not the C-terminal truncated FAAP24 mutant (N150) binds to ICL DNA. Biotinylated control DNA or ICL-DNA (100 ng) were attached to streptavidin-coated beads and incubated with purified His-tagged WT FAAP24 or N150 FAAP24. (C) Immunoblot showing that RPA loading to ICL DNA was decreased in FAAP24-depleted nuclear extracts. Nuclear extracts (100 μg) derived from HeLa scramble or FAAP24 shRNA cells were incubated with biotinylated control DNA or ICL-DNA (100 ng) respectively. The DNA-bound FAAP24, RPA2 and KU70 were detected. (D) Proposed working model for the role of FANCM/FAAP24 in the ICL-induced checkpoint response.
    Figure Legend Snippet: RPA binding to ICL DNA is dependent on FAAP24 (A) Migration of ICL DNA on a denaturing gel. (B) Immunoblot showing that FAAP24 wild-type (WT) but not the C-terminal truncated FAAP24 mutant (N150) binds to ICL DNA. Biotinylated control DNA or ICL-DNA (100 ng) were attached to streptavidin-coated beads and incubated with purified His-tagged WT FAAP24 or N150 FAAP24. (C) Immunoblot showing that RPA loading to ICL DNA was decreased in FAAP24-depleted nuclear extracts. Nuclear extracts (100 μg) derived from HeLa scramble or FAAP24 shRNA cells were incubated with biotinylated control DNA or ICL-DNA (100 ng) respectively. The DNA-bound FAAP24, RPA2 and KU70 were detected. (D) Proposed working model for the role of FANCM/FAAP24 in the ICL-induced checkpoint response.

    Techniques Used: Recombinase Polymerase Amplification, Binding Assay, Migration, Mutagenesis, Incubation, Purification, Derivative Assay, shRNA

    4) Product Images from "Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry"

    Article Title: Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh198

    MALDI-TOF mass spectra of the DNA extension products from four simultaneous primer extension reactions using dUTP-PC-Biotin before and after photocleavage. ( A ) Before UV irradiation. The extension products were purified by solid phase capture and released for MS analysis by using formamide to denature the biotin–streptavidin binding. The double charged ion peaks are indicated by asterisks. ( B ) After 10 min irradiation (340 nm) of the DNA fragments captured on the streptavidin-coated beads, photocleaved DNA products are released without biotin, and detected in the solution.
    Figure Legend Snippet: MALDI-TOF mass spectra of the DNA extension products from four simultaneous primer extension reactions using dUTP-PC-Biotin before and after photocleavage. ( A ) Before UV irradiation. The extension products were purified by solid phase capture and released for MS analysis by using formamide to denature the biotin–streptavidin binding. The double charged ion peaks are indicated by asterisks. ( B ) After 10 min irradiation (340 nm) of the DNA fragments captured on the streptavidin-coated beads, photocleaved DNA products are released without biotin, and detected in the solution.

    Techniques Used: Irradiation, Purification, Mass Spectrometry, Binding Assay

    MALDI-TOF mass spectra of the DNA extension product generated from dUTP-PC-Biotin before and after photocleavage. ( A ) Before UV irradiation. The extension product was purified by solid phase capture and released for MS analysis by using formamide to denature the biotin–streptavidin binding. ( B ) After 10 min irradiation (340 nm) of the DNA fragments captured on the streptavidin-coated beads, the photocleaved DNA fragment 7 is released without biotin, and detected in the solution.
    Figure Legend Snippet: MALDI-TOF mass spectra of the DNA extension product generated from dUTP-PC-Biotin before and after photocleavage. ( A ) Before UV irradiation. The extension product was purified by solid phase capture and released for MS analysis by using formamide to denature the biotin–streptavidin binding. ( B ) After 10 min irradiation (340 nm) of the DNA fragments captured on the streptavidin-coated beads, the photocleaved DNA fragment 7 is released without biotin, and detected in the solution.

    Techniques Used: Generated, Irradiation, Purification, Mass Spectrometry, Binding Assay

    Polymerase extension reaction using dUTP-PC-Biotin as a substrate and photocleavage of DNA fragments containing dU-PC-Biotin on a solid surface. DNA polymerase incorporates dUTP-PC-Biotin and ddGTP to generate the DNA fragment 6 . Photocleavage by near-UV light (340 nm) of the DNA fragment 6 captured on streptavidin-coated beads produces DNA fragment 7 , while the PC-Biotin moiety stays on the solid surface of the beads.
    Figure Legend Snippet: Polymerase extension reaction using dUTP-PC-Biotin as a substrate and photocleavage of DNA fragments containing dU-PC-Biotin on a solid surface. DNA polymerase incorporates dUTP-PC-Biotin and ddGTP to generate the DNA fragment 6 . Photocleavage by near-UV light (340 nm) of the DNA fragment 6 captured on streptavidin-coated beads produces DNA fragment 7 , while the PC-Biotin moiety stays on the solid surface of the beads.

    Techniques Used:

    5) Product Images from "Local palmitoylation cycles define activity-regulated postsynaptic subdomains"

    Article Title: Local palmitoylation cycles define activity-regulated postsynaptic subdomains

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201302071

    DHHC2 directly nucleates PSD-95 assembly at the plasma membrane through local palmitoylation. (A) HEK293T cells were cotransfected with a bi-cistronic RUSH vector containing streptavidin-Ii (Str-ER Hook) and streptavidin-binding peptide (SBP)-GFP-DHHC2 as well as PSD-95-mCherry. Synchronized release of DHHC2 from the ER was induced by the addition of biotin with or without 2-BP. Arrowheads denote signals at the plasma membrane. Bar, 10 µm. (B) Kymograph analysis. The fluorescence intensities of GFP and mCherry were measured along red lines in A. White arrows indicate the timing when DHHC2 arrived at the plasma membrane. Black arrows indicate the position of the plasma membrane (at 90 min). CS, inactive DHHC2. Bar, 2.5 µm.
    Figure Legend Snippet: DHHC2 directly nucleates PSD-95 assembly at the plasma membrane through local palmitoylation. (A) HEK293T cells were cotransfected with a bi-cistronic RUSH vector containing streptavidin-Ii (Str-ER Hook) and streptavidin-binding peptide (SBP)-GFP-DHHC2 as well as PSD-95-mCherry. Synchronized release of DHHC2 from the ER was induced by the addition of biotin with or without 2-BP. Arrowheads denote signals at the plasma membrane. Bar, 10 µm. (B) Kymograph analysis. The fluorescence intensities of GFP and mCherry were measured along red lines in A. White arrows indicate the timing when DHHC2 arrived at the plasma membrane. Black arrows indicate the position of the plasma membrane (at 90 min). CS, inactive DHHC2. Bar, 2.5 µm.

    Techniques Used: Plasmid Preparation, Binding Assay, Fluorescence

    6) Product Images from "Histone modifications influence mediator interactions with chromatin"

    Article Title: Histone modifications influence mediator interactions with chromatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr551

    Assay of Mediator binding to biotinylated synthetic histone tail peptides using streptavidin conjugated magnetic beads. ( A and B ) Wild-type Mediator (~6 nM) was incubated with histone tail peptides (~2 µM) (Input). After incubation of the input with streptavidin beads, the beads were washed, and bound Mediator and peptide eluted from the beads with SDS page loading buffer (Elution). Western blotting using the specified percent of the total input and elution samples and antibodies against subunits from different structural modules of Mediator [α-Med14(Rgr1), α-Med1, α-Med18(Srb5) and α-Med8], were used to analyze the input and elution fractions. ( C ) Wild-type Mediator (~1.5 nM) was incubated with histone tail peptides (~2 µM) (Input). After incubation of the input with streptavidin beads, the beads were washed, and bound Mediator and peptide eluted from the beads with SDS page loading buffer (Elution). Western blotting using the specified percent of the total input and elution samples, and antibodies against subunits from different structural modules of Mediator [α-Med14(Rgr1), α-Med1, and α-Med18(Srb5)], were used to analyze the input and elution fractions. ( D ) Order of affinity of wild-type Mediator for different histone tail peptides.
    Figure Legend Snippet: Assay of Mediator binding to biotinylated synthetic histone tail peptides using streptavidin conjugated magnetic beads. ( A and B ) Wild-type Mediator (~6 nM) was incubated with histone tail peptides (~2 µM) (Input). After incubation of the input with streptavidin beads, the beads were washed, and bound Mediator and peptide eluted from the beads with SDS page loading buffer (Elution). Western blotting using the specified percent of the total input and elution samples and antibodies against subunits from different structural modules of Mediator [α-Med14(Rgr1), α-Med1, α-Med18(Srb5) and α-Med8], were used to analyze the input and elution fractions. ( C ) Wild-type Mediator (~1.5 nM) was incubated with histone tail peptides (~2 µM) (Input). After incubation of the input with streptavidin beads, the beads were washed, and bound Mediator and peptide eluted from the beads with SDS page loading buffer (Elution). Western blotting using the specified percent of the total input and elution samples, and antibodies against subunits from different structural modules of Mediator [α-Med14(Rgr1), α-Med1, and α-Med18(Srb5)], were used to analyze the input and elution fractions. ( D ) Order of affinity of wild-type Mediator for different histone tail peptides.

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

    7) Product Images from "Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats"

    Article Title: Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2016.0167

    Aptamer Ec3(31) mediated E. coli pull-down. Streptavidin coated magnetic beads complexed with Ec3(31)-biotin was used to pull-down E. coli from various concentrations of sample preparations and further cultured on LB plate. Colonies were counted and represented on the Y axis. N40 down primer and SQ2 mutant sequences were used as controls. Results are represented as mean ± SD of 3 independent experiments.
    Figure Legend Snippet: Aptamer Ec3(31) mediated E. coli pull-down. Streptavidin coated magnetic beads complexed with Ec3(31)-biotin was used to pull-down E. coli from various concentrations of sample preparations and further cultured on LB plate. Colonies were counted and represented on the Y axis. N40 down primer and SQ2 mutant sequences were used as controls. Results are represented as mean ± SD of 3 independent experiments.

    Techniques Used: Magnetic Beads, Cell Culture, Mutagenesis

    8) Product Images from "Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax"

    Article Title: Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax

    Journal: Journal of Virology

    doi: 10.1128/JVI.00186-06

    CARM1 is recruited to the HTLV-1 PICs in the presence of Tax. HTLV-1 PICs were assembled by incubating biotinylated HTLV-1 templates with HeLa nuclear extracts (ext) in the absence or presence of the His 6 -Tax WT or mutant (del 151-204) and then purified with streptavidin-coated magnetic beads. The protein components of the purified PICs were analyzed by Western blotting with anti-Tax (A), -CARM1 (B), -CREB (C), or -p300 (D) antibodies. DNA-bio, biotinylated DNA.
    Figure Legend Snippet: CARM1 is recruited to the HTLV-1 PICs in the presence of Tax. HTLV-1 PICs were assembled by incubating biotinylated HTLV-1 templates with HeLa nuclear extracts (ext) in the absence or presence of the His 6 -Tax WT or mutant (del 151-204) and then purified with streptavidin-coated magnetic beads. The protein components of the purified PICs were analyzed by Western blotting with anti-Tax (A), -CARM1 (B), -CREB (C), or -p300 (D) antibodies. DNA-bio, biotinylated DNA.

    Techniques Used: Mutagenesis, Purification, Magnetic Beads, Western Blot

    9) Product Images from "High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting"

    Article Title: High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135278

    Abdurin binding assays. Affinity matured binders to EphA2, selected by CIS display, were tested in end-point titration ELISA assays in 96-well plates coated with hEphA2-Fc (a), mEphA2-Fc (b) or strepavidin (c). The purified matured Abdurin clones D2, G7, B6 and B11 were tested alongside the purified non-matured parental EphA2 binding clones selected by phage display, E10par and H3par, as detected by anti-FLAG M2-HRP conjugated antibody. Binding of Abdurin binders and the truncated wild-type human CH2 (shWTCH2) to CHO cells transfected with human EphA2 (d) or to non-transfected cells (e), was assessed by FACS analysis.
    Figure Legend Snippet: Abdurin binding assays. Affinity matured binders to EphA2, selected by CIS display, were tested in end-point titration ELISA assays in 96-well plates coated with hEphA2-Fc (a), mEphA2-Fc (b) or strepavidin (c). The purified matured Abdurin clones D2, G7, B6 and B11 were tested alongside the purified non-matured parental EphA2 binding clones selected by phage display, E10par and H3par, as detected by anti-FLAG M2-HRP conjugated antibody. Binding of Abdurin binders and the truncated wild-type human CH2 (shWTCH2) to CHO cells transfected with human EphA2 (d) or to non-transfected cells (e), was assessed by FACS analysis.

    Techniques Used: Binding Assay, Titration, Enzyme-linked Immunosorbent Assay, Purification, Clone Assay, Transfection, FACS

    10) Product Images from "DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry"

    Article Title: DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry

    Journal: Nucleic Acids Research

    doi:

    Scheme used to purify DNA-sequencing fragments for analysis by MS. False stops, excess primers and salts are eliminated from the reaction by capturing all correctly terminated DNA-sequencing fragments with streptavidin-coated magnetic beads. The purified fragments are then cleaved from the beads with 98% formamide for analysis by MALDI-TOF MS.
    Figure Legend Snippet: Scheme used to purify DNA-sequencing fragments for analysis by MS. False stops, excess primers and salts are eliminated from the reaction by capturing all correctly terminated DNA-sequencing fragments with streptavidin-coated magnetic beads. The purified fragments are then cleaved from the beads with 98% formamide for analysis by MALDI-TOF MS.

    Techniques Used: DNA Sequencing, Mass Spectrometry, Magnetic Beads, Purification

    11) Product Images from "Galectins control mTOR in response to endomembrane damage"

    Article Title: Galectins control mTOR in response to endomembrane damage

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.03.009

    Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on streptavidin-beads analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .
    Figure Legend Snippet: Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on streptavidin-beads analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .

    Techniques Used: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Incubation, Isolation, GST Pulldown Assay, In Vitro, Binding Assay, Negative Control

    12) Product Images from "Borrelia burgdorferi transcriptome in the central nervous system of non-human primates"

    Article Title: Borrelia burgdorferi transcriptome in the central nervous system of non-human primates

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

    doi: 10.1073/pnas.2432412100

    Schematic representation of DECAL. ( A ) Construction of Bb-CAL. A ZAP II B. burgdorferi genomic library was screened with radioactively labeled B. burgdorferi rRNA probes and hybridizing clones discarded. The nonribosomal clones were pooled and restriction digested with EcoR V and Sma I. The 200- to 2,000-bp fragments were gel-purified, ligated to PCR adapters, and PCR-amplified. ( B ) Positive selection and amplification. Total RNA isolated from heart and CNS of B. burgdorferi -infected NHPs was reverse transcribed in the presence of biotin-dATP. The biotinylated cDNA samples were hybridized to Bb-CAL under stringent conditions. The cDNA-Bb-CAL hybrids were bound to streptavidin-coated magnetic beads and then washed to remove unhybridized Bb-CAL. ( C ) The bound genomic Bb-CAL (representing the B. burgdorferi transcripts in the CNS/heart) was eluted by boiling and PCR-amplified. The PCR products were radioactively labeled and hybridized to a replicate genomic array.
    Figure Legend Snippet: Schematic representation of DECAL. ( A ) Construction of Bb-CAL. A ZAP II B. burgdorferi genomic library was screened with radioactively labeled B. burgdorferi rRNA probes and hybridizing clones discarded. The nonribosomal clones were pooled and restriction digested with EcoR V and Sma I. The 200- to 2,000-bp fragments were gel-purified, ligated to PCR adapters, and PCR-amplified. ( B ) Positive selection and amplification. Total RNA isolated from heart and CNS of B. burgdorferi -infected NHPs was reverse transcribed in the presence of biotin-dATP. The biotinylated cDNA samples were hybridized to Bb-CAL under stringent conditions. The cDNA-Bb-CAL hybrids were bound to streptavidin-coated magnetic beads and then washed to remove unhybridized Bb-CAL. ( C ) The bound genomic Bb-CAL (representing the B. burgdorferi transcripts in the CNS/heart) was eluted by boiling and PCR-amplified. The PCR products were radioactively labeled and hybridized to a replicate genomic array.

    Techniques Used: Labeling, Clone Assay, Purification, Polymerase Chain Reaction, Amplification, Selection, Isolation, Infection, Magnetic Beads

    13) Product Images from "A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination"

    Article Title: A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx571

    PRP19 assembles with RFWD3 on RPA–ssDNA in response to DNA damage and promotes RPA ubiquitylation. ( A ) PRP19 and RFWD3 depletion perturb DNA damage-induced RPA ubiquitylation. Cells were transfected with siRNAs targeting either PRP19 or RFWD3 and a vector expressing His 6 -tagged ubiquitin, treated or not with CPT and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. ( B ) Cells were transfected with SFB- (S-protein, FLAG, streptavidin-binding peptide) GFP or SFB-RFWD3 vectors and streptavidin pulldown of SFB-tagged proteins in untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( C ) Cells were transfected with SFB-GFP or SFB-PRP19 vectors and streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( D ) Cells were transfected with SFB-GFP or SFB-PRP19 and myc-RFWD3 vectors. Streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed and the indicated proteins were immunoblotted. ( E ) HeLa cells transfected with an SFB-PRP19 vector and pre-sensitized with BrdU were UV laser microirradiated. Immunofluorescence against endogenous γ-H2AX, RFWD3 and FLAG epitope was subsequently performed to monitor RFWD3 and PRP19 accrual at damage sites.
    Figure Legend Snippet: PRP19 assembles with RFWD3 on RPA–ssDNA in response to DNA damage and promotes RPA ubiquitylation. ( A ) PRP19 and RFWD3 depletion perturb DNA damage-induced RPA ubiquitylation. Cells were transfected with siRNAs targeting either PRP19 or RFWD3 and a vector expressing His 6 -tagged ubiquitin, treated or not with CPT and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. ( B ) Cells were transfected with SFB- (S-protein, FLAG, streptavidin-binding peptide) GFP or SFB-RFWD3 vectors and streptavidin pulldown of SFB-tagged proteins in untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( C ) Cells were transfected with SFB-GFP or SFB-PRP19 vectors and streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( D ) Cells were transfected with SFB-GFP or SFB-PRP19 and myc-RFWD3 vectors. Streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed and the indicated proteins were immunoblotted. ( E ) HeLa cells transfected with an SFB-PRP19 vector and pre-sensitized with BrdU were UV laser microirradiated. Immunofluorescence against endogenous γ-H2AX, RFWD3 and FLAG epitope was subsequently performed to monitor RFWD3 and PRP19 accrual at damage sites.

    Techniques Used: Recombinase Polymerase Amplification, Transfection, Plasmid Preparation, Expressing, Cycling Probe Technology, Binding Assay, Isolation, Immunofluorescence, FLAG-tag

    DNA-damage-induced RPA phosphorylation promotes its ubiquitylation. ( A ) HeLa cell lines stably expressing HA-tagged WT or Ala10 RPA32 mutants were transfected with a vector expressing SFB-PRP19. Cells were then treated with CPT 1 μM for 4 h, lysed and SFB-PRP19 and its interactors were isolated using streptavidin-associated beads. ( B ) Stable HeLa cell lines expressing the indicated HA-tagged RPA32 constructs were transfected with an siRNA targeting the 3′ untranslated region for the RPA32 mRNA. 72 h later, cells were treated with 1 μM CPT for 2 h, lysed and the indicated proteins were detected using specific antibodies. ( C ) Alternatively, cells transfected as in (B) were microirradiated and processed for immunofluorescence to examine PRP19 recruitment to laser stripes. ( D ) Histogram representing the recruitment of endogenous PRP19 to RPA32 stripes after laser microirradiation. The error bars correspond to biological triplicate experiments. At least 100 microirradiated cells were examined for each replicate. ( E ) Cells were transfected with vectors expressing His 6 -tagged ubiquitin and myc-tagged WT or Ala10 RPA32 mutants and treated with 1 μM CPT for 3 h. Ubiquitylated proteins were isolated by denaturing Ni-NTA pulldown.
    Figure Legend Snippet: DNA-damage-induced RPA phosphorylation promotes its ubiquitylation. ( A ) HeLa cell lines stably expressing HA-tagged WT or Ala10 RPA32 mutants were transfected with a vector expressing SFB-PRP19. Cells were then treated with CPT 1 μM for 4 h, lysed and SFB-PRP19 and its interactors were isolated using streptavidin-associated beads. ( B ) Stable HeLa cell lines expressing the indicated HA-tagged RPA32 constructs were transfected with an siRNA targeting the 3′ untranslated region for the RPA32 mRNA. 72 h later, cells were treated with 1 μM CPT for 2 h, lysed and the indicated proteins were detected using specific antibodies. ( C ) Alternatively, cells transfected as in (B) were microirradiated and processed for immunofluorescence to examine PRP19 recruitment to laser stripes. ( D ) Histogram representing the recruitment of endogenous PRP19 to RPA32 stripes after laser microirradiation. The error bars correspond to biological triplicate experiments. At least 100 microirradiated cells were examined for each replicate. ( E ) Cells were transfected with vectors expressing His 6 -tagged ubiquitin and myc-tagged WT or Ala10 RPA32 mutants and treated with 1 μM CPT for 3 h. Ubiquitylated proteins were isolated by denaturing Ni-NTA pulldown.

    Techniques Used: Recombinase Polymerase Amplification, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Cycling Probe Technology, Isolation, Construct, Immunofluorescence

    The PRP19 WD40 domain contains an electropositive surface that mediates its damage-induced interaction with the RPA complex. ( A and B ) Electrostatic surface potential of the WD40 repeats domain of human PRP19 identifies two major electropositive surfaces (blue). The mutated positively charged residues in the mPocket1 and mPocket2 PRP19 constructs are shown in magenta. ( C ) Cells were transfected with WT SFB-PRP19 or the indicated mutants and treated with 1 μM CPT 24 h later. SFB-PRP19 constructs and their interactors were isolated using streptavidin-associated beads and immunoblotted with the indicated antibodies.
    Figure Legend Snippet: The PRP19 WD40 domain contains an electropositive surface that mediates its damage-induced interaction with the RPA complex. ( A and B ) Electrostatic surface potential of the WD40 repeats domain of human PRP19 identifies two major electropositive surfaces (blue). The mutated positively charged residues in the mPocket1 and mPocket2 PRP19 constructs are shown in magenta. ( C ) Cells were transfected with WT SFB-PRP19 or the indicated mutants and treated with 1 μM CPT 24 h later. SFB-PRP19 constructs and their interactors were isolated using streptavidin-associated beads and immunoblotted with the indicated antibodies.

    Techniques Used: Recombinase Polymerase Amplification, Construct, Transfection, Cycling Probe Technology, Isolation

    RPA32 ubiquitylation is regulated by PI3K-like kinases. (A) Cells were transfected with a vector expressing Strep-HA-tagged ubiquitin, pre-treated for 1 h with VE-821 (ATRi, 10 μM), KU55933 (ATMi, 10 uM), NU7441 (DNAPKi, 2 μM) or a combination of all three inhibitors, treated with 1 μM CPT for 4 h and lysed under denaturing conditions. Strep-Tactin pulldown was performed to isolate ubiquitylated proteins. ( B ) Cell transfection with an SFB-PRP19 vector were treated as in A and streptavidin pulldown was performed to isolate PRP19 along with its interactors. Controls for the efficiency of the inhibitor treatments are provided in ( S2A ). ( C and D ) U2OS cells were pre-sensitized with 10 μM BrdU, treated with the indicated inhibitors along with DRB for 1 h and damaged by UV-laser microirradiation. Recruitment of PRP19 and RPA32 was monitored 2 h after damage by immunofluorescence.
    Figure Legend Snippet: RPA32 ubiquitylation is regulated by PI3K-like kinases. (A) Cells were transfected with a vector expressing Strep-HA-tagged ubiquitin, pre-treated for 1 h with VE-821 (ATRi, 10 μM), KU55933 (ATMi, 10 uM), NU7441 (DNAPKi, 2 μM) or a combination of all three inhibitors, treated with 1 μM CPT for 4 h and lysed under denaturing conditions. Strep-Tactin pulldown was performed to isolate ubiquitylated proteins. ( B ) Cell transfection with an SFB-PRP19 vector were treated as in A and streptavidin pulldown was performed to isolate PRP19 along with its interactors. Controls for the efficiency of the inhibitor treatments are provided in ( S2A ). ( C and D ) U2OS cells were pre-sensitized with 10 μM BrdU, treated with the indicated inhibitors along with DRB for 1 h and damaged by UV-laser microirradiation. Recruitment of PRP19 and RPA32 was monitored 2 h after damage by immunofluorescence.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Cycling Probe Technology, Immunofluorescence

    RPA32 is phosphorylated and ubiquitylated in response to DNA damage that targets active replication forks. ( A ) Cells were transfected with a vector expressing His 6 -tagged ubiquitin and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. The indicated proteins were detected with specific antibodies. ( B ) Cells were transfected with a vector expressing Strep-HA ubiquitin and treated with 1 or 5 μM CPT, 10 γ IR, 4 mM HU or 50 J/m 2 UV for 4 h. Ubiquitylated proteins were isolated by Strep-Tactin pulldown under denaturing conditions. ( C ) Cells transfected as in (B) were treated with 1 μM CPT for the indicated times and total RPA32 or ( D ) phosphorylated RPA32 species were detected using specific antibodies after Strep-Tactin pulldown. ( E ) A stable HEK293T cell line expressing SFB-PRP19 was treated with CPT 1 μM for the indicated times. SFB-PRP19 and its interactors were isolated using streptavidin-associated beads.
    Figure Legend Snippet: RPA32 is phosphorylated and ubiquitylated in response to DNA damage that targets active replication forks. ( A ) Cells were transfected with a vector expressing His 6 -tagged ubiquitin and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. The indicated proteins were detected with specific antibodies. ( B ) Cells were transfected with a vector expressing Strep-HA ubiquitin and treated with 1 or 5 μM CPT, 10 γ IR, 4 mM HU or 50 J/m 2 UV for 4 h. Ubiquitylated proteins were isolated by Strep-Tactin pulldown under denaturing conditions. ( C ) Cells transfected as in (B) were treated with 1 μM CPT for the indicated times and total RPA32 or ( D ) phosphorylated RPA32 species were detected using specific antibodies after Strep-Tactin pulldown. ( E ) A stable HEK293T cell line expressing SFB-PRP19 was treated with CPT 1 μM for the indicated times. SFB-PRP19 and its interactors were isolated using streptavidin-associated beads.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Cycling Probe Technology, Isolation

    14) Product Images from "Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition"

    Article Title: Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.3.601

    Fig. 1. ( A ) Schematic representation of the construction of the double-stranded AFM substrate. The different DNA strands of the 5′ biotinylated PCR fragments (indicated by small filled circles) are isolated using streptavidin-coated magnetic beads (indicated by large filled circles). The 297 nt top strand is added to the immobilized 1032 nt bottom strand together with the 50 nt phosphorylated ‘AFM-chol’ oligo containing the cholesterol adduct. After hybridization and ligation, the incomplete top strand is extended by T 7 polymerase to produce a completely double-stranded fragment. Digestion with Sma I yields a 1020 bp DNA fragment containing a single cholesterol adduct at position 324 in the top strand. ( B ) DNA sequence of the 50 bp S1 substrate. The cholesterol adduct (Chol) is introduced at position 27 (X) in the top strand. The incision positions are indicated with arrows.
    Figure Legend Snippet: Fig. 1. ( A ) Schematic representation of the construction of the double-stranded AFM substrate. The different DNA strands of the 5′ biotinylated PCR fragments (indicated by small filled circles) are isolated using streptavidin-coated magnetic beads (indicated by large filled circles). The 297 nt top strand is added to the immobilized 1032 nt bottom strand together with the 50 nt phosphorylated ‘AFM-chol’ oligo containing the cholesterol adduct. After hybridization and ligation, the incomplete top strand is extended by T 7 polymerase to produce a completely double-stranded fragment. Digestion with Sma I yields a 1020 bp DNA fragment containing a single cholesterol adduct at position 324 in the top strand. ( B ) DNA sequence of the 50 bp S1 substrate. The cholesterol adduct (Chol) is introduced at position 27 (X) in the top strand. The incision positions are indicated with arrows.

    Techniques Used: Polymerase Chain Reaction, Isolation, Magnetic Beads, Hybridization, Ligation, Sequencing

    15) Product Images from "Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection"

    Article Title: Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection

    Journal: Scientific Reports

    doi: 10.1038/srep12090

    ( A ) Construction of a random DNA oligonucleotide library. Genomic DNA was size-fractionated, size-selected, and PCR amplified to generate the genomic library. Two hybrid primers (hyb) consisting of nine random nucleotides (red) followed by specific sequences (light and dark blue, fix primers) were used to specifically amplify the library. After synthesis, reaction products are size-selected by gel electrophoresis. The fraction that contains library fragments of the desired size range is eluted and amplified by PCR with the fix-primer pair. The T7 promoter (dashed line) for transcription of the library into RNA is incorporated in the fix-FOR primer sequence, according to Zimmermann et al .5 with minor modifications. ( B ) Schematic representation of the genomic SELEX, according to Stoltenburg et al .1 with modifications. Streptavidin-coated magnetic beads coupled to biotinylated PCA3 were used to select RNA aptamers, forming a target:aptamer complex. The SELEX procedure consisted of eight cycles of selection steps: binding, washing, elution, amplification and purification. A newly enriched pool of selected oligonucleotides was generated by preparation of the relevant ssDNA for in vitro transcription. The enriched aptamer pool was cloned, and several individual aptamers were characterized by sequencing.
    Figure Legend Snippet: ( A ) Construction of a random DNA oligonucleotide library. Genomic DNA was size-fractionated, size-selected, and PCR amplified to generate the genomic library. Two hybrid primers (hyb) consisting of nine random nucleotides (red) followed by specific sequences (light and dark blue, fix primers) were used to specifically amplify the library. After synthesis, reaction products are size-selected by gel electrophoresis. The fraction that contains library fragments of the desired size range is eluted and amplified by PCR with the fix-primer pair. The T7 promoter (dashed line) for transcription of the library into RNA is incorporated in the fix-FOR primer sequence, according to Zimmermann et al .5 with minor modifications. ( B ) Schematic representation of the genomic SELEX, according to Stoltenburg et al .1 with modifications. Streptavidin-coated magnetic beads coupled to biotinylated PCA3 were used to select RNA aptamers, forming a target:aptamer complex. The SELEX procedure consisted of eight cycles of selection steps: binding, washing, elution, amplification and purification. A newly enriched pool of selected oligonucleotides was generated by preparation of the relevant ssDNA for in vitro transcription. The enriched aptamer pool was cloned, and several individual aptamers were characterized by sequencing.

    Techniques Used: Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis, Sequencing, Magnetic Beads, Selection, Binding Assay, Purification, Generated, In Vitro, Clone Assay

    16) Product Images from "A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer’s Disease"

    Article Title: A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer’s Disease

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2019.00310

    Biotinylated PNR502 localizes to aggregates in AM141 worms and is used to recover drug-adherent proteins. (A) Structure of biotinyl-PNR502. (B,C) AM141 adult worms were either untreated (B) or treated 26 h (C) with 10-μM PNR502 and then fed Alexa594-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were captured with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope. Q40::YFP is displayed as green, and Alexa594 as red fluorescence. Yellow fluorescence in (C) indicates the superposition of Q40::YFP with PNR502-Alexa594. (D) Caudal hippocampi, from normal age-matched controls (AMC) or AD patients (pools of three per group), were lysed and incubated 2 h at 4°C with 5-μM PNR502 or biotinyl-PNR502. M , size markers; lanes 1–4, proteins recovered from: 1 , unmodified PNR502; 2 , biotinyl-PNR502; 3 , equivalent portion of flow-through for unmodified PNR502; 4 , flow-through from biotinyl-PNR502.
    Figure Legend Snippet: Biotinylated PNR502 localizes to aggregates in AM141 worms and is used to recover drug-adherent proteins. (A) Structure of biotinyl-PNR502. (B,C) AM141 adult worms were either untreated (B) or treated 26 h (C) with 10-μM PNR502 and then fed Alexa594-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were captured with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope. Q40::YFP is displayed as green, and Alexa594 as red fluorescence. Yellow fluorescence in (C) indicates the superposition of Q40::YFP with PNR502-Alexa594. (D) Caudal hippocampi, from normal age-matched controls (AMC) or AD patients (pools of three per group), were lysed and incubated 2 h at 4°C with 5-μM PNR502 or biotinyl-PNR502. M , size markers; lanes 1–4, proteins recovered from: 1 , unmodified PNR502; 2 , biotinyl-PNR502; 3 , equivalent portion of flow-through for unmodified PNR502; 4 , flow-through from biotinyl-PNR502.

    Techniques Used: Fluorescence, Inverted Microscopy, Incubation, Flow Cytometry

    17) Product Images from "Characterization of plasma labile heme in hemolytic conditions"

    Article Title: Characterization of plasma labile heme in hemolytic conditions

    Journal: The FEBS journal

    doi: 10.1111/febs.14192

    Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P
    Figure Legend Snippet: Characterization of labile heme in plasma following acute hemolysis. (A) Number of RBC in C57BL/6 mice receiving Phenylhydrazine (PHX) and control (CTRL) mice receiving PBS. (B) Heme concentration in the plasma of C57BL/6 mice receiving phenylhydrazine. (C) Correlation between circulating RBC numbers (data from A) and heme concentration in plasma (data from B). (D) Concentration of bioavailable heme in plasma of C57BL/6 mice receiving phenylhydrazine, quantified by a heme reporter assay [ 31 ]. (E) Correlation between circulating RBC numbers (data from A) and concentration of bioavailable heme in plasma (data from D). (F) Soluble hemin quantified by a sandwich ELISA in which the sdAbs 1A6 and 2H7 are used to capture and reveal heme, respectively. (G) Detection of soluble heme versus heme bound to HPX using the same sdAb-based ELISA as in (F). Note that heme bound to HPX is not detected by ELISA. (H) A pull-down assay using streptavidin-beads to capture heme-biotin. The sdAb 2H10 bound to heme-biotin was added to HPX at 1/6 SdAb/HPX molar ratio. Streptavidin-beads pulled down the sdAb 2H10 as well as HPX bound to heme-biotin, demonstrating that HPX can bind heme-bound to sdAb 2H10. This is consistent with the higher affinity of HPX toward heme as compared to the sdAb 2H10. Coomassie-based stain of 15% SDS/PAGE gel loaded with streptavidin-beads used to pull-down heme-biotin from different reaction mixtures. Grey arrowheads indicate the molecular weight of the protein ladder (NZYColour Protein Marker II, Nzytech ® ) in kDa loaded in the first lane of the gel. Gel is representative of two independent experiments with similar trend. (I) Plasma HBC 1/2 in C57BL/6 mice receiving phenylhydrazine. Circles in A, B, C, D, E, and I correspond to individual mice. Red dash line represents mean ± STD. * P

    Techniques Used: Mouse Assay, Concentration Assay, Reporter Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Pull Down Assay, Staining, SDS Page, Molecular Weight, Marker

    Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.
    Figure Legend Snippet: Analysis of heme binding by SdAbs. (A) SdAbs bound to biotinylated-heme in solution were pooled-down using streptavidin (SA) beads and detected by western blot using anti-HA mAb. Input was measured by Coomassie-based stain. (B) UV-Visible spectra of hemin. Soret region at approximately 364 and 383 nm and a CT band at 622 nm are shown, representative of three independent experiments. (C) UV-visible spectra of sdAb 2H10, 1A6 and 2H7 bound to heme at different concentrations. Soret (412 nm), Q 1 (530 nm), Q 0 (565 nm), and CT (624 nm) bands are highlighted. (D) Far UV CD spectra of sdAb 2H10 in the apo (black) and heme-bound (red) forms. Shift from 212 to 218 is due to heme-driven conformational rearrangement of the sdAb secondary structure. The inset shows the Soret region, with the appearance of the 412 nm band, due to heme binding to the sdAb. (E) ATR FTIR absorption spectra (top) and second derivative (bottom) of sdAb 2H10 in the apo (black) and heme-bound (red) forms in the amide I region (1700–1610 cm −1 ), showing structural modification upon heme coordination. (F) High frequency Resonance Raman spectra of hemin and sdAb 2H10 bound to hemin, obtained with 413 nm excitation.

    Techniques Used: Binding Assay, Western Blot, Staining, Modification

    18) Product Images from "Single Bead Affinity Detection (SINBAD) for the Analysis of Protein-Protein Interactions"

    Article Title: Single Bead Affinity Detection (SINBAD) for the Analysis of Protein-Protein Interactions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002061

    SINBAD for the detection of protein-DNA interactions. (A) his-TRF2 was immobilized on Ni-NTA beads and incubated with hybridized 5′-biotinylated DNA oligos containing three repeats of TTAGGG (red), a mutant TTACGG or ssTTAGG as indicated. Bound DNA was visualized with streptavidin-coated QDs655. (B)
    Figure Legend Snippet: SINBAD for the detection of protein-DNA interactions. (A) his-TRF2 was immobilized on Ni-NTA beads and incubated with hybridized 5′-biotinylated DNA oligos containing three repeats of TTAGGG (red), a mutant TTACGG or ssTTAGG as indicated. Bound DNA was visualized with streptavidin-coated QDs655. (B)

    Techniques Used: Incubation, Mutagenesis

    Monitoring protein interactions on a single affinity chromatography bead. (A) 1 µM recombinant his-TAP-tagged Importin β(wt) was immobilized on 5 ul magnetic Ni-NTA beads for 20 min. After the addition of 5 nM CdSe-ZnS core shell QD655 beads were washed and isolated with a magnet. Beads were recovered in 2 ul PBS, mounted on a glass slide and the bead surface was imaged by confocal microscopy. (B) Schematic illustration of the RanGTP (gray) dissociation of Importin β (red) from Importin α (light blue). (c) QD585 cross-linked to NLS peptide (blue), QD705 cross-linked to reverse NLS (SLN) (magenta), streptavidin-coated QD655-TAP-Importin β(wt) (red) and streptavidin-coated QD605-TAP-Importin β(ΔN44) (green) were incubated with his-Importin α coated Ni-NTA beads in the presence of buffer (control), 20 mM NLS or SLN peptides or 10 mM RanQ69L.
    Figure Legend Snippet: Monitoring protein interactions on a single affinity chromatography bead. (A) 1 µM recombinant his-TAP-tagged Importin β(wt) was immobilized on 5 ul magnetic Ni-NTA beads for 20 min. After the addition of 5 nM CdSe-ZnS core shell QD655 beads were washed and isolated with a magnet. Beads were recovered in 2 ul PBS, mounted on a glass slide and the bead surface was imaged by confocal microscopy. (B) Schematic illustration of the RanGTP (gray) dissociation of Importin β (red) from Importin α (light blue). (c) QD585 cross-linked to NLS peptide (blue), QD705 cross-linked to reverse NLS (SLN) (magenta), streptavidin-coated QD655-TAP-Importin β(wt) (red) and streptavidin-coated QD605-TAP-Importin β(ΔN44) (green) were incubated with his-Importin α coated Ni-NTA beads in the presence of buffer (control), 20 mM NLS or SLN peptides or 10 mM RanQ69L.

    Techniques Used: Affinity Chromatography, Recombinant, Isolation, Confocal Microscopy, Incubation

    Using SINBAD as pull-down method with increased sensitivity. (A) Approx. 10 Ni-NTA beads were incubated with reticulocyte lysates containing his-Importin α or his- Importin α together with in vitro translated TAP-Importin β(wt) in the presence or absence of excess Importin α. QD655 were added, beads were isolated and imaged. (B) his-Nup160 was translated in reticulocyte lysates and incubated with equal volume (2 ul) of in vitro translated Nup133-TAP, Nup107-TAP, Nup96-TAP, Nup85-TAP, Nup43-TAP, Nup37-TAP, Seh1-TAP, Sec13-TAP and ELYS-TAP for 30 min. Approx. 5 Ni-NTA beads were added together with streptavidin-coated QDs655 and imaged by confocal microscopy on the bead surface. (C) Schematic illustration of SINBAD for the pull-down of endogenous proteins. (D) Hypotonic cell lysates corresponding to 500 or 50 cells were incubated with Ni-NTA beads coated with his-RanQ69L. Bound endogenous Importin β was visualized as described in (C). (E) Random 10 µm 2 areas were imaged and fluorescence intensity at 655 nm was plotted. n = 20.
    Figure Legend Snippet: Using SINBAD as pull-down method with increased sensitivity. (A) Approx. 10 Ni-NTA beads were incubated with reticulocyte lysates containing his-Importin α or his- Importin α together with in vitro translated TAP-Importin β(wt) in the presence or absence of excess Importin α. QD655 were added, beads were isolated and imaged. (B) his-Nup160 was translated in reticulocyte lysates and incubated with equal volume (2 ul) of in vitro translated Nup133-TAP, Nup107-TAP, Nup96-TAP, Nup85-TAP, Nup43-TAP, Nup37-TAP, Seh1-TAP, Sec13-TAP and ELYS-TAP for 30 min. Approx. 5 Ni-NTA beads were added together with streptavidin-coated QDs655 and imaged by confocal microscopy on the bead surface. (C) Schematic illustration of SINBAD for the pull-down of endogenous proteins. (D) Hypotonic cell lysates corresponding to 500 or 50 cells were incubated with Ni-NTA beads coated with his-RanQ69L. Bound endogenous Importin β was visualized as described in (C). (E) Random 10 µm 2 areas were imaged and fluorescence intensity at 655 nm was plotted. n = 20.

    Techniques Used: Incubation, In Vitro, Isolation, Confocal Microscopy, Fluorescence

    19) Product Images from "The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain"

    Article Title: The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1468

    Nrd1 binds preferentially to CTD-Ser5P. ( a ) Binding to four repeat CTD peptides in vitro . Unmodified, Ser2P, Ser5P or Ser2P/Ser5P peptides were immobilized on streptavidin-conjugated magnetic beads and incubated with 5 µg of recombinant Nrd1 (rNrd1), 10 ng of TAP-purified yeast Nrd1 complex (yNRD1) or 500 µg of whole-cell extract from an Rtt103-hemagglutinin (HA) strain (YSB815). Bound proteins were eluted, separated by SDS-PAGE and detected by immunoblotting using either anti-Nrd1 or anti-HA antibodies. Recombinant Rtt103 also specifically bound to CTD-Ser2P (not shown). ( b ) Nrd1 6–151 was titrated with fluorescently labeled CTD-Ser5P (two repeats) and binding was measured by fluorescence anisotropy (black triangles; ref.-FAM, 5,6-carboxyfluorescein labeled reference). The same experiment was then done in the presence of competing unlabeled CTD-Ser2P (circles), CTDSer5P (white triangles) or CTD-Ser2P/Ser5P (diamonds). Equilibrium dissociation constants ( K d ) were calculated from the best fit to the data. ( c ) Nrd1 is associated with Ser5-phosphorylated Pol II in vivo . Nrd1 was purified via the TAP tag, and the phosphorylation status of the associated polymerase was monitored by immunoblotting using anti-CTD (8WG16), anti-Ser2P (H5), anti-Ser5P (H14) or an antibody that can recognize both Ser2P and Ser5P (B3) 9 . ( d ) Ctk1 kinase is not required for recruitment of Nrd1 to genes in vivo . Cross-linked chromatin was prepared from Nrd1-TAP–containing cells that were wild-type (WT) or deleted (Δctk1) for the CTK1 gene. Following precipitation with IgG agarose, chromatin was amplified with primers across the snR33 locus, as diagrammed below. Immunoprecipitated samples (IP) were compared against input chromatin (Input) and quantified (right). The upper band in each lane is the snR33 product and the lower band is a nontranscribed control region. Similar results were obtained for the PMA1 and ADH1 genes (not shown).
    Figure Legend Snippet: Nrd1 binds preferentially to CTD-Ser5P. ( a ) Binding to four repeat CTD peptides in vitro . Unmodified, Ser2P, Ser5P or Ser2P/Ser5P peptides were immobilized on streptavidin-conjugated magnetic beads and incubated with 5 µg of recombinant Nrd1 (rNrd1), 10 ng of TAP-purified yeast Nrd1 complex (yNRD1) or 500 µg of whole-cell extract from an Rtt103-hemagglutinin (HA) strain (YSB815). Bound proteins were eluted, separated by SDS-PAGE and detected by immunoblotting using either anti-Nrd1 or anti-HA antibodies. Recombinant Rtt103 also specifically bound to CTD-Ser2P (not shown). ( b ) Nrd1 6–151 was titrated with fluorescently labeled CTD-Ser5P (two repeats) and binding was measured by fluorescence anisotropy (black triangles; ref.-FAM, 5,6-carboxyfluorescein labeled reference). The same experiment was then done in the presence of competing unlabeled CTD-Ser2P (circles), CTDSer5P (white triangles) or CTD-Ser2P/Ser5P (diamonds). Equilibrium dissociation constants ( K d ) were calculated from the best fit to the data. ( c ) Nrd1 is associated with Ser5-phosphorylated Pol II in vivo . Nrd1 was purified via the TAP tag, and the phosphorylation status of the associated polymerase was monitored by immunoblotting using anti-CTD (8WG16), anti-Ser2P (H5), anti-Ser5P (H14) or an antibody that can recognize both Ser2P and Ser5P (B3) 9 . ( d ) Ctk1 kinase is not required for recruitment of Nrd1 to genes in vivo . Cross-linked chromatin was prepared from Nrd1-TAP–containing cells that were wild-type (WT) or deleted (Δctk1) for the CTK1 gene. Following precipitation with IgG agarose, chromatin was amplified with primers across the snR33 locus, as diagrammed below. Immunoprecipitated samples (IP) were compared against input chromatin (Input) and quantified (right). The upper band in each lane is the snR33 product and the lower band is a nontranscribed control region. Similar results were obtained for the PMA1 and ADH1 genes (not shown).

    Techniques Used: Binding Assay, In Vitro, Magnetic Beads, Incubation, Recombinant, Purification, SDS Page, Labeling, Fluorescence, In Vivo, Amplification, Immunoprecipitation

    20) Product Images from "Enrichment of meiotic recombination hotspot sequences by avidin capture technology"

    Article Title: Enrichment of meiotic recombination hotspot sequences by avidin capture technology

    Journal: Gene

    doi: 10.1016/j.gene.2012.12.042

    Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.
    Figure Legend Snippet: Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker

    Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI . Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).
    Figure Legend Snippet: Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI . Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).

    Techniques Used: Labeling, Sequencing, Fluorescence

    Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.
    Figure Legend Snippet: Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker

    21) Product Images from "Galectins control mTOR in response to endomembrane damage"

    Article Title: Galectins control mTOR in response to endomembrane damage

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.03.009

    Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on streptavidin-beads analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .
    Figure Legend Snippet: Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on streptavidin-beads analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .

    Techniques Used: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Incubation, Isolation, GST Pulldown Assay, In Vitro, Binding Assay, Negative Control

    22) Product Images from "A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer’s Disease"

    Article Title: A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer’s Disease

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2019.00310

    Biotinylated PNR502 localizes to aggregates in AM141 worms and is used to recover drug-adherent proteins. (A) Structure of biotinyl-PNR502. (B,C) AM141 adult worms were either untreated (B) or treated 26 h (C) with 10-μM PNR502 and then fed Alexa594-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were captured with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope. Q40::YFP is displayed as green, and Alexa594 as red fluorescence. Yellow fluorescence in (C) indicates the superposition of Q40::YFP with PNR502-Alexa594. (D) Caudal hippocampi, from normal age-matched controls (AMC) or AD patients (pools of three per group), were lysed and incubated 2 h at 4°C with 5-μM PNR502 or biotinyl-PNR502. M , size markers; lanes 1–4, proteins recovered from: 1 , unmodified PNR502; 2 , biotinyl-PNR502; 3 , equivalent portion of flow-through for unmodified PNR502; 4 , flow-through from biotinyl-PNR502.
    Figure Legend Snippet: Biotinylated PNR502 localizes to aggregates in AM141 worms and is used to recover drug-adherent proteins. (A) Structure of biotinyl-PNR502. (B,C) AM141 adult worms were either untreated (B) or treated 26 h (C) with 10-μM PNR502 and then fed Alexa594-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were captured with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope. Q40::YFP is displayed as green, and Alexa594 as red fluorescence. Yellow fluorescence in (C) indicates the superposition of Q40::YFP with PNR502-Alexa594. (D) Caudal hippocampi, from normal age-matched controls (AMC) or AD patients (pools of three per group), were lysed and incubated 2 h at 4°C with 5-μM PNR502 or biotinyl-PNR502. M , size markers; lanes 1–4, proteins recovered from: 1 , unmodified PNR502; 2 , biotinyl-PNR502; 3 , equivalent portion of flow-through for unmodified PNR502; 4 , flow-through from biotinyl-PNR502.

    Techniques Used: Fluorescence, Inverted Microscopy, Incubation, Flow Cytometry

    23) Product Images from "A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling"

    Article Title: A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling

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

    doi: 10.1073/pnas.252624099

    Reduced amounts of Notch 3 R142C at the cell surface. Cell surface proteins of HEK 293 stable cell lines expressing either the mNotch 3 or the mNotch 3 R142C receptor were biotinylated. Five percent of the protein extract was removed before the pull down with magnetic beads coupled to streptavidin. The remaining 95% of the extracted proteins was subjected to the streptavidin pull down (B) and followed by SDS/PAGE and Western blot analysis by using the 5E1 antibody along with 5% of total protein extract (T). Note that the ratio between the pulled-down receptor (B) and the total amount of expressed receptor (T) is lower for mNotch 3 R142C compared with wild-type mNotch 3. The 210-kDa immunoreactive band that is present in all pulled-down fractions suggests that both the vast majority of wild-type and mNotch 3 R142C receptors are expressed as S1-cleaved bipartite proteins at the cell surface. The Western blot is from one representative experiment, whereas the 210:280-kDa ratios are mean ± SD from three independent experiments.
    Figure Legend Snippet: Reduced amounts of Notch 3 R142C at the cell surface. Cell surface proteins of HEK 293 stable cell lines expressing either the mNotch 3 or the mNotch 3 R142C receptor were biotinylated. Five percent of the protein extract was removed before the pull down with magnetic beads coupled to streptavidin. The remaining 95% of the extracted proteins was subjected to the streptavidin pull down (B) and followed by SDS/PAGE and Western blot analysis by using the 5E1 antibody along with 5% of total protein extract (T). Note that the ratio between the pulled-down receptor (B) and the total amount of expressed receptor (T) is lower for mNotch 3 R142C compared with wild-type mNotch 3. The 210-kDa immunoreactive band that is present in all pulled-down fractions suggests that both the vast majority of wild-type and mNotch 3 R142C receptors are expressed as S1-cleaved bipartite proteins at the cell surface. The Western blot is from one representative experiment, whereas the 210:280-kDa ratios are mean ± SD from three independent experiments.

    Techniques Used: Stable Transfection, Expressing, Magnetic Beads, SDS Page, Western Blot

    24) Product Images from "RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription"

    Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription

    Journal: Gene Expression

    doi:

    Characterization of RNAs associated with the Pur factor. The RNA content of the heparin-agarose fraction of the Pur factor was analyzed by 3′ labeling with [ 32 P]Cp, electrophoresis through an 8% polyacrylamide sequencing gel, and visualized by autoradiography. Lane T: total display of RNA molecules obtained by phenol extraction of the fraction containing the Pur factor (fraction 14 of the heparin-agarose column). Lanes P and S correspond to RNAs extracted from the same fraction incubated with a biotinylated PUR oligonucleotide and reacted with streptavidin-coated magnetic beads. Lane P is the Pur factor fraction pelleted with the beads and lane S is the corresponding supernatant. Lane C is a control similar to P, using beads uncoupled to the PUR oligonucleotide. All samples were separated by electrophoresis through an 8% polyacrylamide sequencing gel and were visualized by autoradiography.
    Figure Legend Snippet: Characterization of RNAs associated with the Pur factor. The RNA content of the heparin-agarose fraction of the Pur factor was analyzed by 3′ labeling with [ 32 P]Cp, electrophoresis through an 8% polyacrylamide sequencing gel, and visualized by autoradiography. Lane T: total display of RNA molecules obtained by phenol extraction of the fraction containing the Pur factor (fraction 14 of the heparin-agarose column). Lanes P and S correspond to RNAs extracted from the same fraction incubated with a biotinylated PUR oligonucleotide and reacted with streptavidin-coated magnetic beads. Lane P is the Pur factor fraction pelleted with the beads and lane S is the corresponding supernatant. Lane C is a control similar to P, using beads uncoupled to the PUR oligonucleotide. All samples were separated by electrophoresis through an 8% polyacrylamide sequencing gel and were visualized by autoradiography.

    Techniques Used: Labeling, Electrophoresis, Sequencing, Autoradiography, Incubation, Magnetic Beads

    25) Product Images from "SDA, a DNA Aptamer Inhibiting E- and P-Selectin Mediated Adhesion of Cancer and Leukemia Cells, the First and Pivotal Step in Transendothelial Migration during Metastasis Formation"

    Article Title: SDA, a DNA Aptamer Inhibiting E- and P-Selectin Mediated Adhesion of Cancer and Leukemia Cells, the First and Pivotal Step in Transendothelial Migration during Metastasis Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093173

    Affinities of selected DNA aptamers to rh E- and rh P-selectin determined via filter retention assays (FRA). DNA was radiolabeled, incubated with increasing amounts of proteins and filtrated through a nitrocellulose membrane. Fractions of bound DNAs were detected via autoradiography and quantified. (A) Recombinant human E-selectin incubated with DNA pool after one (▴) and 17 (•) SELEX rounds. (B) Aptamer SDA incubated with rh E-selectin (•, K d ≈ 87 nM), rh P-selectin (▪, K d ≈ 84 nM), or streptavidin (▴) as a control. A control DNA did neither bind to human E- (▾) nor P-selectin (♦).
    Figure Legend Snippet: Affinities of selected DNA aptamers to rh E- and rh P-selectin determined via filter retention assays (FRA). DNA was radiolabeled, incubated with increasing amounts of proteins and filtrated through a nitrocellulose membrane. Fractions of bound DNAs were detected via autoradiography and quantified. (A) Recombinant human E-selectin incubated with DNA pool after one (▴) and 17 (•) SELEX rounds. (B) Aptamer SDA incubated with rh E-selectin (•, K d ≈ 87 nM), rh P-selectin (▪, K d ≈ 84 nM), or streptavidin (▴) as a control. A control DNA did neither bind to human E- (▾) nor P-selectin (♦).

    Techniques Used: Incubation, Autoradiography, Recombinant

    26) Product Images from "Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library"

    Article Title: Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149774

    Schematic of the building blocks for DNA construction. Streptavidin-coated magnetic beads were used as solid support for dsDNA synthesis and oligo fragments were ligated to the building block one at a time.
    Figure Legend Snippet: Schematic of the building blocks for DNA construction. Streptavidin-coated magnetic beads were used as solid support for dsDNA synthesis and oligo fragments were ligated to the building block one at a time.

    Techniques Used: Magnetic Beads, Blocking Assay

    Schematic diagram for the proposed dsDNA synthesis. The overall procedure for dsDNA synthesis is composed of three processes: annealing, binding of streptavidin coated magnetic beads to biotinylated oligos, and ligation.
    Figure Legend Snippet: Schematic diagram for the proposed dsDNA synthesis. The overall procedure for dsDNA synthesis is composed of three processes: annealing, binding of streptavidin coated magnetic beads to biotinylated oligos, and ligation.

    Techniques Used: Binding Assay, Magnetic Beads, Ligation

    27) Product Images from "Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome"

    Article Title: Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.6b00115

    Visualizing the N 3 QM modified proteins in HT-29 cells: (A) lysates from HT-29 cells treated with DMSO, azido- p NO-ASA (10 µM), azido- p Br-ASA (10 µM), clicked to biotin, and applied on to the streptavidin-coated magnetic beads to separate the QM-modified proteins from the unmodified. All fractions were run on SDS–PAGE gel electrophoresis and stained with Coomassie brilliant blue. (B) Lysates from HT-29 cells treated with DMSO, azido- p NO-ASA (10 µM), and azido- p Br-ASA (10 µM), with or without p NO-ASA (10 µM) or p Br-ASA (10 µM) pretreatments, clicked to biotin, and subjected to Western blotting with the use of antibiotin-HRP for visual characterization of the modified proteins. Representative Western blotting analysis from three independent experiments are shown.
    Figure Legend Snippet: Visualizing the N 3 QM modified proteins in HT-29 cells: (A) lysates from HT-29 cells treated with DMSO, azido- p NO-ASA (10 µM), azido- p Br-ASA (10 µM), clicked to biotin, and applied on to the streptavidin-coated magnetic beads to separate the QM-modified proteins from the unmodified. All fractions were run on SDS–PAGE gel electrophoresis and stained with Coomassie brilliant blue. (B) Lysates from HT-29 cells treated with DMSO, azido- p NO-ASA (10 µM), and azido- p Br-ASA (10 µM), with or without p NO-ASA (10 µM) or p Br-ASA (10 µM) pretreatments, clicked to biotin, and subjected to Western blotting with the use of antibiotin-HRP for visual characterization of the modified proteins. Representative Western blotting analysis from three independent experiments are shown.

    Techniques Used: Modification, Magnetic Beads, SDS Page, Nucleic Acid Electrophoresis, Staining, Western Blot

    28) Product Images from "Efficient preparation of internally modified single-molecule constructs using nicking enzymes"

    Article Title: Efficient preparation of internally modified single-molecule constructs using nicking enzymes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1004

    Schematic representation of the internal labeling method. ( a ) A DNA sequence which incorporates five equally spaced BbvCI recognition sites (black triangles) is nicked only at one of the two strands using either the nicking enzyme Nt.BbvCI or Nb.BbvCI. This results in the formation of short 15–16 bases long fragments. Denaturation and subsequent hybridization, in the presence of a DNA strand (shown in red) that is complementary to the resulting 63 bp gap and that carries the desired internal modifications (e.g. two or six biotins as depicted), lead to an efficient replacement of the original fragments with the labeled fragment. The spacing between the internal modifications of 10–11 bp ensures that they extrude in the same direction from the DNA. ( b ) Model of a streptavidin tetramer bound to an internally biotinylated DNA molecule (Streptavidin PDB id: 1MK5; the bound monomer is illustrated as a yellow ribbon while for the other subunits the surface representation was used. DNA PDB id: 2BNA). The attachment of the streptavidin tetramer to only one of the biotins was arbitrarily chosen.
    Figure Legend Snippet: Schematic representation of the internal labeling method. ( a ) A DNA sequence which incorporates five equally spaced BbvCI recognition sites (black triangles) is nicked only at one of the two strands using either the nicking enzyme Nt.BbvCI or Nb.BbvCI. This results in the formation of short 15–16 bases long fragments. Denaturation and subsequent hybridization, in the presence of a DNA strand (shown in red) that is complementary to the resulting 63 bp gap and that carries the desired internal modifications (e.g. two or six biotins as depicted), lead to an efficient replacement of the original fragments with the labeled fragment. The spacing between the internal modifications of 10–11 bp ensures that they extrude in the same direction from the DNA. ( b ) Model of a streptavidin tetramer bound to an internally biotinylated DNA molecule (Streptavidin PDB id: 1MK5; the bound monomer is illustrated as a yellow ribbon while for the other subunits the surface representation was used. DNA PDB id: 2BNA). The attachment of the streptavidin tetramer to only one of the biotins was arbitrarily chosen.

    Techniques Used: Labeling, Sequencing, Hybridization

    Site-specific attachment of Q-dots to internally biotinylated DNA. ( a ) Band-shift assay of Q-dot binding to DNA. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb. (Lane 1) pNLrep after digestion with BamHI, PspOMI and Nt.BbvCI and internal biotinylation with oligomer biotinx2 ( Figure 2 a). (Lane 2) Sample from Lane 1 with 5-fold molar excess of streptavidin coated Q-dots added. (Lane 3) Sample containing Q-dots only. Symbols on the right side indicate Q-dots (yellow spheres), the short biotinylated fragment (red line) and the long non-biotinylated fragment (blue line). ( b and c ) AFM images of Q-dots bound to DNA. The colour scale corresponds to a height-range of 1 nm, and the scale bar corresponds to 100 nm. ( d ) Histogram of the Q-dot position measured from the nearest DNA end. The expected Q-dot position at 920 bp (310 nm) (blue dashed line) is within the double confidence interval (light grey band) of the experimentally determined mean (290 ± 20 nm, red dashed line).
    Figure Legend Snippet: Site-specific attachment of Q-dots to internally biotinylated DNA. ( a ) Band-shift assay of Q-dot binding to DNA. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb. (Lane 1) pNLrep after digestion with BamHI, PspOMI and Nt.BbvCI and internal biotinylation with oligomer biotinx2 ( Figure 2 a). (Lane 2) Sample from Lane 1 with 5-fold molar excess of streptavidin coated Q-dots added. (Lane 3) Sample containing Q-dots only. Symbols on the right side indicate Q-dots (yellow spheres), the short biotinylated fragment (red line) and the long non-biotinylated fragment (blue line). ( b and c ) AFM images of Q-dots bound to DNA. The colour scale corresponds to a height-range of 1 nm, and the scale bar corresponds to 100 nm. ( d ) Histogram of the Q-dot position measured from the nearest DNA end. The expected Q-dot position at 920 bp (310 nm) (blue dashed line) is within the double confidence interval (light grey band) of the experimentally determined mean (290 ± 20 nm, red dashed line).

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

    Internal modification and religation efficiencies. ( a ) Polyacrylamide gel electrophoresis of DNA samples in the course of the internal labeling procedure. (Lane 1) pNLrep after digestion with MluI and AatII yielding a 0.8-kb fragment (red line) that carries the region to be replaced as well as a 0.4-kb (light blue line) and a 5-kbp fragment (dark blue line). (Lane 2) pNLrep after simultaneous digestion with MluI, AatII and Nt.BbvCI. The nicking of the 0.8-kb fragment (represented by the fragmented red line) can be seen as a slight mobility decrease. (Lane 3) Sample from lane 2 after column purification, which leads to gap formation within the 0.8-kb fragment causing a large mobility alteration (gapped red line). (Lane 4) Sample from lane 2 after the replacement reaction with oligo biotinx2, during which the 0.8-kb fragment becomes internally biotinylated, and subsequent column purification. The inserted oligo is stably bound and therefore displays the same mobility as the nicked fragment in lane 2. (Lane 5) Sample from lane 4 with > 10-fold molar excess of streptavidin added. (Lane 6) Pulldown assay with sample from lane 4 (see ‘Materials and Methods' section). (Lanes 0, 7) 100 bp step DNA ladder, starting at 400 bp with an additional 517 bp band. ( b ) (Lane 1) pNLrep. (Lane 2) pNLrep after nicking and internal biotinylation with oligo biotinx2. (Lane 3) Sample from lane 2 after ligation. (Lane 4) pNLrep after internal biotinylation with 5′-phosphorylated biotinx2 oligo and religation. (Lane 5) pNLrep after nicking with Nt.BbvCI and religation. Positions of supercoiled, nicked and linearized plasmid species are indicated by corresponding symbols at the right side. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb.
    Figure Legend Snippet: Internal modification and religation efficiencies. ( a ) Polyacrylamide gel electrophoresis of DNA samples in the course of the internal labeling procedure. (Lane 1) pNLrep after digestion with MluI and AatII yielding a 0.8-kb fragment (red line) that carries the region to be replaced as well as a 0.4-kb (light blue line) and a 5-kbp fragment (dark blue line). (Lane 2) pNLrep after simultaneous digestion with MluI, AatII and Nt.BbvCI. The nicking of the 0.8-kb fragment (represented by the fragmented red line) can be seen as a slight mobility decrease. (Lane 3) Sample from lane 2 after column purification, which leads to gap formation within the 0.8-kb fragment causing a large mobility alteration (gapped red line). (Lane 4) Sample from lane 2 after the replacement reaction with oligo biotinx2, during which the 0.8-kb fragment becomes internally biotinylated, and subsequent column purification. The inserted oligo is stably bound and therefore displays the same mobility as the nicked fragment in lane 2. (Lane 5) Sample from lane 4 with > 10-fold molar excess of streptavidin added. (Lane 6) Pulldown assay with sample from lane 4 (see ‘Materials and Methods' section). (Lanes 0, 7) 100 bp step DNA ladder, starting at 400 bp with an additional 517 bp band. ( b ) (Lane 1) pNLrep. (Lane 2) pNLrep after nicking and internal biotinylation with oligo biotinx2. (Lane 3) Sample from lane 2 after ligation. (Lane 4) pNLrep after internal biotinylation with 5′-phosphorylated biotinx2 oligo and religation. (Lane 5) pNLrep after nicking with Nt.BbvCI and religation. Positions of supercoiled, nicked and linearized plasmid species are indicated by corresponding symbols at the right side. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb.

    Techniques Used: Modification, Polyacrylamide Gel Electrophoresis, Labeling, Purification, Stable Transfection, Ligation, Plasmid Preparation

    ssDNA to dsDNA ligation at nicking-enzyme-generated overhangs. ( a ) Schematic representation of overhang generation. A BbvCI recognition site (blue letters) was incorporated near the DNA end in such a way that nicking with Nt.BbvCI generates a 10-bp fragment at the 5′-end. ( b ) Agarose gel of DNA fragments, ligation products and streptavidin-induced band shifts. The biotinylated 40-bp hairpin, the 430-bp dsDNA handle and streptavidin are represented by a blue, red and green symbol, respectively. `Lig.' indicates where a ligation for 1 h at room temperature was carried out. Positions of the reaction products are marked at the right side. (Lanes 1–5) reaction products for the 4 nt overhang generated by BstXI. (Lanes 6–10) reaction products for the 10 nt overhang generated by Nt.BbvCI. The lane in the middle is a 100-bp size marker ladder with the shortest fragment starting at 100-bp and 100-bp size difference between all subsequent fragments (and an additional band at 517 bp). The success of the ssDNA to dsDNA ligation was confirmed by the streptavidin-induced band-shift, in which the desired product specifically shifted only in the case where a 10 nt 3′-overhang had been used. ( c ) Magnetic tweezers experiment with the generated hairpin construct. The molecule was held at the critical force where the closed and the opened states of the hairpin (as illustrated by the sketches) were nearly equally populated. The change in height between the two states was ≈38 nm as expected for a 40-nt hairpin.
    Figure Legend Snippet: ssDNA to dsDNA ligation at nicking-enzyme-generated overhangs. ( a ) Schematic representation of overhang generation. A BbvCI recognition site (blue letters) was incorporated near the DNA end in such a way that nicking with Nt.BbvCI generates a 10-bp fragment at the 5′-end. ( b ) Agarose gel of DNA fragments, ligation products and streptavidin-induced band shifts. The biotinylated 40-bp hairpin, the 430-bp dsDNA handle and streptavidin are represented by a blue, red and green symbol, respectively. `Lig.' indicates where a ligation for 1 h at room temperature was carried out. Positions of the reaction products are marked at the right side. (Lanes 1–5) reaction products for the 4 nt overhang generated by BstXI. (Lanes 6–10) reaction products for the 10 nt overhang generated by Nt.BbvCI. The lane in the middle is a 100-bp size marker ladder with the shortest fragment starting at 100-bp and 100-bp size difference between all subsequent fragments (and an additional band at 517 bp). The success of the ssDNA to dsDNA ligation was confirmed by the streptavidin-induced band-shift, in which the desired product specifically shifted only in the case where a 10 nt 3′-overhang had been used. ( c ) Magnetic tweezers experiment with the generated hairpin construct. The molecule was held at the critical force where the closed and the opened states of the hairpin (as illustrated by the sketches) were nearly equally populated. The change in height between the two states was ≈38 nm as expected for a 40-nt hairpin.

    Techniques Used: Ligation, Generated, Agarose Gel Electrophoresis, Marker, Electrophoretic Mobility Shift Assay, Construct

    29) Product Images from "miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif"

    Article Title: miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00259

    MAZ binds to pre-miR-1207 and -647 in the nucleus. (A) Schematic illustration of the workflow of the RNA pull-down assay. Biotinylated pre-miRNA were conjugated to streptavidin-coated beads and used as bait to retrieve binding proteins from pre-cleared nuclear lysate. (B) Design of pre-miR oligos for electrophoretic mobility shift assay (EMSA). “MAZ” labels indicate putative MAZ binding sites. Red labels indicate where native sequences were edited to achieve the 80 nt length limit. (C) Predicted hairpin structures of native and edited EMSA pre-miRs. Bulge loop features from the original predictions were retained in the new sequences. Oligo design and structure prediction was done using Geneious software. (D) Western blot detection of RNA pulldown assay. MAZ was detected at 55 kDa. (E) EMSA demonstrating shift of pre-miRs by addition of nuclear lysate. Me1a1 oligo was used as a competitive inhibitor to binding, which reversed the observed shift. (F) Western-EMSA (WEMSA) shows the presence of MAZ in the shifted band.
    Figure Legend Snippet: MAZ binds to pre-miR-1207 and -647 in the nucleus. (A) Schematic illustration of the workflow of the RNA pull-down assay. Biotinylated pre-miRNA were conjugated to streptavidin-coated beads and used as bait to retrieve binding proteins from pre-cleared nuclear lysate. (B) Design of pre-miR oligos for electrophoretic mobility shift assay (EMSA). “MAZ” labels indicate putative MAZ binding sites. Red labels indicate where native sequences were edited to achieve the 80 nt length limit. (C) Predicted hairpin structures of native and edited EMSA pre-miRs. Bulge loop features from the original predictions were retained in the new sequences. Oligo design and structure prediction was done using Geneious software. (D) Western blot detection of RNA pulldown assay. MAZ was detected at 55 kDa. (E) EMSA demonstrating shift of pre-miRs by addition of nuclear lysate. Me1a1 oligo was used as a competitive inhibitor to binding, which reversed the observed shift. (F) Western-EMSA (WEMSA) shows the presence of MAZ in the shifted band.

    Techniques Used: Pull Down Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Software, Western Blot

    30) Product Images from "Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods"

    Article Title: Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods

    Journal: Bioorganic & medicinal chemistry

    doi: 10.1016/j.bmc.2011.03.012

    HTS for RPA–Rad9 interactions. RPA bound to biotinylated-ssDNA in a streptavidin coated 384-well plate was mixed with the indicated compounds, A, B, C, and D (D represents NSC15520), and percent inhibition was normalized to reactions that did
    Figure Legend Snippet: HTS for RPA–Rad9 interactions. RPA bound to biotinylated-ssDNA in a streptavidin coated 384-well plate was mixed with the indicated compounds, A, B, C, and D (D represents NSC15520), and percent inhibition was normalized to reactions that did

    Techniques Used: Recombinase Polymerase Amplification, Inhibition

    Purification and interaction of recombinant RPA and GST–Rad9. (A) Proteins were separated by SDS–PAGE and stained with Coomassie blue. (B) Pulldown of Rad9 with ssDNA bound RPA using streptavidin-linked magnetic beads. Lane 1 does not
    Figure Legend Snippet: Purification and interaction of recombinant RPA and GST–Rad9. (A) Proteins were separated by SDS–PAGE and stained with Coomassie blue. (B) Pulldown of Rad9 with ssDNA bound RPA using streptavidin-linked magnetic beads. Lane 1 does not

    Techniques Used: Purification, Recombinant, Recombinase Polymerase Amplification, SDS Page, Staining, Magnetic Beads

    31) Product Images from "A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells"

    Article Title: A medium hyperglycosylated podocalyxin enables noninvasive and quantitative detection of tumorigenic human pluripotent stem cells

    Journal: Scientific Reports

    doi: 10.1038/srep04069

    The GlycoStem test discriminates undifferentiated cells from differentiated cells. Biotinylated rBC2LCN (0.1 μg/well) was immobilized on streptavidin-coated 96-well microtiter plates at 37°C for 1 h. Cell culture supernatants of MEF and 253G1 hiPSCs with or without retinoic acid (RA) treatments for 15 days were incubated at 37°C for 1 h. After washing, HRP-labeled rABA (0.1 μg/mL, 50 μL) was overlayed at 37°C for 1 h. After washing, absorbance at 450 nm was then detected. Absorbance at 450 nm of the control cell culture media was subtracted from the values obtained from the cell culture supernatants. Data are shown as mean ± SD of triplicate samples.
    Figure Legend Snippet: The GlycoStem test discriminates undifferentiated cells from differentiated cells. Biotinylated rBC2LCN (0.1 μg/well) was immobilized on streptavidin-coated 96-well microtiter plates at 37°C for 1 h. Cell culture supernatants of MEF and 253G1 hiPSCs with or without retinoic acid (RA) treatments for 15 days were incubated at 37°C for 1 h. After washing, HRP-labeled rABA (0.1 μg/mL, 50 μL) was overlayed at 37°C for 1 h. After washing, absorbance at 450 nm was then detected. Absorbance at 450 nm of the control cell culture media was subtracted from the values obtained from the cell culture supernatants. Data are shown as mean ± SD of triplicate samples.

    Techniques Used: Cell Culture, Incubation, Labeling

    32) Product Images from "Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution"

    Article Title: Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000055

    The T-array protocol. (A) During development, VDJ recombination causes enormous variability in TCRβ chain by randomly selecting different combinations of 23 V, 2 D, and 13 J gene segments, by nucleotide insertion ( ), and by nucleotide deletion from V ( ), D, and J ( ) genes. This results in a diversity of an estimated 10 6 different β chains per individual. (B) N-deletion causes shortening of the Vβ and Jβ segments. The number of nucleotides deleted from Vβ and Jβ germline DNA is limited. N-deletion of 192 published TCRβ mRNAs was determined. The figure shows the cumulative percentage of CDR3βs for the number of nucleotides deleted. TCRβ's with n nucleotides deleted represent approximately 10% of the repertoire if n = 0 to 6, and 5%, if n = 7 to 9. (C) The T-array protocol: (C1) cDNA from T-cells is generated. (C2) CDR3β regions are PCR amplified using biotinylated Vβ-specific ( ) or Vβ-generic primers (not shown here). (C3) Biotinylated strands are removed after alkaline denaturation using streptavidin-coated beads. (c4) Single-strands of polyclonal TCRs are aliquoted and hybridized to fluorescently labeled annealers ( ) complementary to the NDN-adjacent end of a Jβ gene. A specific number of Jβ-gene nucleotides (n) is deleted for each annealer, accounting for N-deletion during the VDJ recombination process. Insert (C4): Each annealer will hybridize to TCRβ rearrangements where n nucleotides are deleted from the Jβ-germline gene segment ( C4A ) or where less than n nucleotides are deleted ( C4B ). (C5) The annealer-hybridized fractions are loaded on universal hexamer arrays for (C6) T-cell-clone-specific ligation and, (C7) subsequently washed, scanned and analyzed.
    Figure Legend Snippet: The T-array protocol. (A) During development, VDJ recombination causes enormous variability in TCRβ chain by randomly selecting different combinations of 23 V, 2 D, and 13 J gene segments, by nucleotide insertion ( ), and by nucleotide deletion from V ( ), D, and J ( ) genes. This results in a diversity of an estimated 10 6 different β chains per individual. (B) N-deletion causes shortening of the Vβ and Jβ segments. The number of nucleotides deleted from Vβ and Jβ germline DNA is limited. N-deletion of 192 published TCRβ mRNAs was determined. The figure shows the cumulative percentage of CDR3βs for the number of nucleotides deleted. TCRβ's with n nucleotides deleted represent approximately 10% of the repertoire if n = 0 to 6, and 5%, if n = 7 to 9. (C) The T-array protocol: (C1) cDNA from T-cells is generated. (C2) CDR3β regions are PCR amplified using biotinylated Vβ-specific ( ) or Vβ-generic primers (not shown here). (C3) Biotinylated strands are removed after alkaline denaturation using streptavidin-coated beads. (c4) Single-strands of polyclonal TCRs are aliquoted and hybridized to fluorescently labeled annealers ( ) complementary to the NDN-adjacent end of a Jβ gene. A specific number of Jβ-gene nucleotides (n) is deleted for each annealer, accounting for N-deletion during the VDJ recombination process. Insert (C4): Each annealer will hybridize to TCRβ rearrangements where n nucleotides are deleted from the Jβ-germline gene segment ( C4A ) or where less than n nucleotides are deleted ( C4B ). (C5) The annealer-hybridized fractions are loaded on universal hexamer arrays for (C6) T-cell-clone-specific ligation and, (C7) subsequently washed, scanned and analyzed.

    Techniques Used: Generated, Polymerase Chain Reaction, Amplification, Labeling, Ligation

    33) Product Images from "circFGFR4 Promotes Differentiation of Myoblasts via Binding miR-107 to Relieve Its Inhibition of Wnt3a"

    Article Title: circFGFR4 Promotes Differentiation of Myoblasts via Binding miR-107 to Relieve Its Inhibition of Wnt3a

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.02.012

    circFGFR4 Binding miR-107 to Promote Cell Differentiation (A) circFGFR4 expression in different tissues from embryonic Qinchuan cattle detected by real-time qPCR. (B) The expression efficiency of pcDNA-circFGFR4 is shown. (C) Bovine primary myoblasts were co-transfected with miR-107 mimic and pCK-circFGFR4-W or pCK-circFGFR4-Mut. Renilla luciferase activity was normalized to the Firefly luciferase activity. (D) qPCR analysis of circFGFR4 level in the streptavidin captured fractions from the bovine primary myoblasts lysates after transfection with 3′ end biotinylated miR-107 or control RNA (NC). (E) Biotin-labeled circRNA was purified and subjected to RNA pull-down assays by incubation with bovine primary myoblasts lysates, followed by qPCR analysis of miR-107 level. (F) The mRNA expression of Wnt3a in primary bovine myoblasts transfected with miR-107 mimic and (or) circFGFR4 for 24 hr was detected by qPCR. (G and H) The expression of MyoG in primary bovine myoblasts was detected by qPCR (G) and western blotting (H). (I) Bovine primary myocytes were transfected with pcDNA-circFGFR4 and (or) miR-107 mimic, and cell differentiation was detected by immunofluorescence (MyHC) and observed under a fluorescence microscope. Values are means ± SEM for three individuals. *p
    Figure Legend Snippet: circFGFR4 Binding miR-107 to Promote Cell Differentiation (A) circFGFR4 expression in different tissues from embryonic Qinchuan cattle detected by real-time qPCR. (B) The expression efficiency of pcDNA-circFGFR4 is shown. (C) Bovine primary myoblasts were co-transfected with miR-107 mimic and pCK-circFGFR4-W or pCK-circFGFR4-Mut. Renilla luciferase activity was normalized to the Firefly luciferase activity. (D) qPCR analysis of circFGFR4 level in the streptavidin captured fractions from the bovine primary myoblasts lysates after transfection with 3′ end biotinylated miR-107 or control RNA (NC). (E) Biotin-labeled circRNA was purified and subjected to RNA pull-down assays by incubation with bovine primary myoblasts lysates, followed by qPCR analysis of miR-107 level. (F) The mRNA expression of Wnt3a in primary bovine myoblasts transfected with miR-107 mimic and (or) circFGFR4 for 24 hr was detected by qPCR. (G and H) The expression of MyoG in primary bovine myoblasts was detected by qPCR (G) and western blotting (H). (I) Bovine primary myocytes were transfected with pcDNA-circFGFR4 and (or) miR-107 mimic, and cell differentiation was detected by immunofluorescence (MyHC) and observed under a fluorescence microscope. Values are means ± SEM for three individuals. *p

    Techniques Used: Binding Assay, Cell Differentiation, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Labeling, Purification, Incubation, Western Blot, Immunofluorescence, Fluorescence, Microscopy

    34) Product Images from "Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs"

    Article Title: Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs

    Journal: Nature Communications

    doi: 10.1038/ncomms11215

    circHIPK3 sponges with miR-124 and inhibits its activity. ( a ) Proliferation assessed using a CCK-8 kit in HEK-293 T cells transfected with nine miRNA mimics or control RNA (20 nM). ( b ) qRT–PCR analysis of circHIPK3 level in the streptavidin captured fractions from the HEK-293 T cell lysates after transfection with 3′-end biotinylated miR-124 or control RNA (NC). ( c ) Co-localization between miR-124 and circHIPK3 was observed (arrowheads) by RNA in situ hybridization in HeLa cells after co-transfection with circHIPK3 and miR-124 expressing vectors. Nuclei were stained with DAPI. Scale bar, 5μm. ( d ) qRT–PCR analysis of IL6R and DLX2 expression in HEK-293 T cells after transfected with si-cHIPK3, miR-124 mimics or miR-124 with circHIPK3 expressing vector (p-cHIPK3). ( e ) Proliferation assessed using a CCK-8 kit in cells transfected with circHIPK3 or miR-124 (10 nM) as indicated. Data in a , b , d are the means±s.e.m. of three experiments. ( f ) qRT–PCR for the abundance of circHIPK3 relative to ACTB and miR-124 relative RNU6B in six human normal tissues. The correlation between circHIPK3 and miR-124 is also shown. * P
    Figure Legend Snippet: circHIPK3 sponges with miR-124 and inhibits its activity. ( a ) Proliferation assessed using a CCK-8 kit in HEK-293 T cells transfected with nine miRNA mimics or control RNA (20 nM). ( b ) qRT–PCR analysis of circHIPK3 level in the streptavidin captured fractions from the HEK-293 T cell lysates after transfection with 3′-end biotinylated miR-124 or control RNA (NC). ( c ) Co-localization between miR-124 and circHIPK3 was observed (arrowheads) by RNA in situ hybridization in HeLa cells after co-transfection with circHIPK3 and miR-124 expressing vectors. Nuclei were stained with DAPI. Scale bar, 5μm. ( d ) qRT–PCR analysis of IL6R and DLX2 expression in HEK-293 T cells after transfected with si-cHIPK3, miR-124 mimics or miR-124 with circHIPK3 expressing vector (p-cHIPK3). ( e ) Proliferation assessed using a CCK-8 kit in cells transfected with circHIPK3 or miR-124 (10 nM) as indicated. Data in a , b , d are the means±s.e.m. of three experiments. ( f ) qRT–PCR for the abundance of circHIPK3 relative to ACTB and miR-124 relative RNU6B in six human normal tissues. The correlation between circHIPK3 and miR-124 is also shown. * P

    Techniques Used: Activity Assay, CCK-8 Assay, Transfection, Quantitative RT-PCR, RNA In Situ Hybridization, Cotransfection, Expressing, Staining, Plasmid Preparation

    35) Product Images from "PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy"

    Article Title: PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008074

    Proposed methodology for PCR-free identification of GMOs by magnetic capture-FCCS. Genomic DNA is isolated from GMOs and then fragmented. Biotin-labeled DNA is hybridized with the 35S promoter region and streptavidin coated-magnetic beads are used to capture the targets from the sample and then washed. The single strand targets are released and hybridized with two fluorophore labeled probes for FCCS detection.
    Figure Legend Snippet: Proposed methodology for PCR-free identification of GMOs by magnetic capture-FCCS. Genomic DNA is isolated from GMOs and then fragmented. Biotin-labeled DNA is hybridized with the 35S promoter region and streptavidin coated-magnetic beads are used to capture the targets from the sample and then washed. The single strand targets are released and hybridized with two fluorophore labeled probes for FCCS detection.

    Techniques Used: Polymerase Chain Reaction, Isolation, Labeling, Magnetic Beads

    36) Product Images from "Biochemical and structural analysis of the interaction between β-amyloid and fibrinogen"

    Article Title: Biochemical and structural analysis of the interaction between β-amyloid and fibrinogen

    Journal: Blood

    doi: 10.1182/blood-2016-03-705228

    Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated
    Figure Legend Snippet: Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated

    Techniques Used: Labeling, Incubation

    37) Product Images from "Vibrio vulnificus quorum-sensing molecule cyclo(Phe-Pro) inhibits RIG-I-mediated antiviral innate immunity"

    Article Title: Vibrio vulnificus quorum-sensing molecule cyclo(Phe-Pro) inhibits RIG-I-mediated antiviral innate immunity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04075-1

    HCV-promoting activity of cFP is mediated through its specific interaction with RIG-I. a , b Immunoblotting analysis of cFP-interacting proteins pull-downed by biotinylated cFP bound to Streptavidin beads ( a ) or by non-modified cFP immobilized onto Sepharose beads ( b ). HEK293T cells, which were non-stimulated ( a ) or stimulated with the indicated RNA ligands ( b ) were used in pull-down experiments. c , d Quantification of IFN-β mRNA ( c ) and HCV genome ( d ) levels in Huh7.5.1 cells transfected with an empty vector or a vector expressing the wild-type RIG-I (RIG-I_WT) or its inactive mutant (RIG-I_K270A). After 18 h, the transfected cells were infected with HCV (JFH1) by incubation for 6 h and incubated further for 24 h in fresh media without or with the indicated peptides (2.5 mM) prior to RT-qPCR analyses. Statistical significance of differences between groups was determined via unpaired two-tailed t -test. * P ≤ 0.05; n.s., not significant
    Figure Legend Snippet: HCV-promoting activity of cFP is mediated through its specific interaction with RIG-I. a , b Immunoblotting analysis of cFP-interacting proteins pull-downed by biotinylated cFP bound to Streptavidin beads ( a ) or by non-modified cFP immobilized onto Sepharose beads ( b ). HEK293T cells, which were non-stimulated ( a ) or stimulated with the indicated RNA ligands ( b ) were used in pull-down experiments. c , d Quantification of IFN-β mRNA ( c ) and HCV genome ( d ) levels in Huh7.5.1 cells transfected with an empty vector or a vector expressing the wild-type RIG-I (RIG-I_WT) or its inactive mutant (RIG-I_K270A). After 18 h, the transfected cells were infected with HCV (JFH1) by incubation for 6 h and incubated further for 24 h in fresh media without or with the indicated peptides (2.5 mM) prior to RT-qPCR analyses. Statistical significance of differences between groups was determined via unpaired two-tailed t -test. * P ≤ 0.05; n.s., not significant

    Techniques Used: Activity Assay, Modification, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Infection, Incubation, Quantitative RT-PCR, Two Tailed Test

    38) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).
    Figure Legend Snippet: DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).

    Techniques Used: Periodic Counter-current Chromatography, SDS Page, Molecular Weight, Marker, Negative Control, Binding Assay, Concentration Assay

    39) Product Images from "The Sulfolobus solfataricus radA paralogue sso0777 is DNA damage inducible and positively regulated by the Sta1 protein"

    Article Title: The Sulfolobus solfataricus radA paralogue sso0777 is DNA damage inducible and positively regulated by the Sta1 protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm782

    SDS–PAGE silver-stained gel showing the pull-down experiment using streptavidin-coated magnetic beads. Magnetic beads were bound to biotinylated DNA from the promoter region of sso0777 (lanes 3 and 4), and incubated with S. solfataricus cell extracts. The FT contains all S. solfataricus proteins which were not bound to the sso0777 promoter; and the bound proteins where eluted (Elution) as described in Materials and Methods section. As a control, the same procedure was carried out using non-biotinylated DNA (lanes 1 and 2). Arrows indicate the two identified proteins, Sso0110 and Sso0048.
    Figure Legend Snippet: SDS–PAGE silver-stained gel showing the pull-down experiment using streptavidin-coated magnetic beads. Magnetic beads were bound to biotinylated DNA from the promoter region of sso0777 (lanes 3 and 4), and incubated with S. solfataricus cell extracts. The FT contains all S. solfataricus proteins which were not bound to the sso0777 promoter; and the bound proteins where eluted (Elution) as described in Materials and Methods section. As a control, the same procedure was carried out using non-biotinylated DNA (lanes 1 and 2). Arrows indicate the two identified proteins, Sso0110 and Sso0048.

    Techniques Used: SDS Page, Staining, Magnetic Beads, Incubation

    40) Product Images from "A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens"

    Article Title: A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens

    Journal: Journal of Immune Based Therapies and Vaccines

    doi: 10.1186/1476-8518-4-2

    Purification of Hsp70 and Hsp70-PCs from MDA-MB-231 cells by affinity chromatography . Hsp70-peptide complexes (Hsp70-PCs) were isolated from whole cell extracts of MDA-MB-231 cells using ADP-Agarose. A . Coomassie-Blue stained SDS-polyacrylamide gel and B : Western blot using anti-Hsp70 antibody. Lane 1: MDA-MB-231 total cell extract (10 μg), Lane 2: Flow-through from an ADP-agarose column (2 μg), Lane 3: Proteins eluted from ADP-agarose column with 3 mM ADP (2 μg). Lane 4: Molecular weight markers. C : ELISA to detect the interaction between biotinylated TMG and DSP peptides and the corresponding phages. Streptavidin-coated paramagnetic beads bound to biotinylated TMG peptide (TMG) or DSP peptide (DSP) were incubated with the M13 phage clones displaying DSP or TMG respectively. As a control, streptavidin-coated beads without the peptides were incubated with M13 phage clone displaying the TMG (TMG negative) or the DSP (DSP negative) peptides alone. All beads were then incubated with anti-M13-HRP antibody. Interactions were detected by absorbance at 405 nm using DAB as a substrate.
    Figure Legend Snippet: Purification of Hsp70 and Hsp70-PCs from MDA-MB-231 cells by affinity chromatography . Hsp70-peptide complexes (Hsp70-PCs) were isolated from whole cell extracts of MDA-MB-231 cells using ADP-Agarose. A . Coomassie-Blue stained SDS-polyacrylamide gel and B : Western blot using anti-Hsp70 antibody. Lane 1: MDA-MB-231 total cell extract (10 μg), Lane 2: Flow-through from an ADP-agarose column (2 μg), Lane 3: Proteins eluted from ADP-agarose column with 3 mM ADP (2 μg). Lane 4: Molecular weight markers. C : ELISA to detect the interaction between biotinylated TMG and DSP peptides and the corresponding phages. Streptavidin-coated paramagnetic beads bound to biotinylated TMG peptide (TMG) or DSP peptide (DSP) were incubated with the M13 phage clones displaying DSP or TMG respectively. As a control, streptavidin-coated beads without the peptides were incubated with M13 phage clone displaying the TMG (TMG negative) or the DSP (DSP negative) peptides alone. All beads were then incubated with anti-M13-HRP antibody. Interactions were detected by absorbance at 405 nm using DAB as a substrate.

    Techniques Used: Purification, Multiple Displacement Amplification, Affinity Chromatography, Isolation, Staining, Western Blot, Flow Cytometry, Molecular Weight, Enzyme-linked Immunosorbent Assay, Incubation, Clone Assay

    Related Articles

    Isolation:

    Article Title: Generation and Nuclear Translocation of Sumoylated Transmembrane Fragment of Cell Adhesion Molecule L1
    Article Snippet: .. For isolation of biotinylated proteins after cell surface biotinylation, streptavidin-conjugated magnetic beads (Pierce) were incubated with cell lysates or cellular subfractions overnight at 4 °C. ..

    Article Title: Surface L-type Ca2+ channel expression levels are increased in aged hippocampus
    Article Snippet: .. Alternate slices from the dorsal half of both left and right hippocampi were exposed to 1 mg mL−1 Sulfo-NHS–SS–biot in-labeling reagent (Pierce) for 30 min before separating each hippocampal region for processing and isolation of surface proteins using streptavidin magnetic beads (Pierce). .. Immunoblotting with p1928, CNC1, and Ab144 antibody was performed after SDS-PAGE separation of total region lysates (input) and biotinylated (surface) fraction proteins.

    Centrifugation:

    Article Title: Carbonic anhydrase-related protein CA10 is an evolutionarily conserved pan-neurexin ligand
    Article Snippet: .. Lysates were cleared by centrifugation at 20,000 × g for 15 min, diluted 1:1 in RIPA, and, following removal of the input fraction, incubated with magnetic streptavidin beads (Pierce) under orbital rotation for 3 h at 4 °C. ..

    Article Title: BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization
    Article Snippet: .. Lysate was clarified by centrifugation and streptavidin magnetic beads (Pierce) were added to each sample and incubated at 4 °C with rotation for 16 h. Beads were washed 4 times with wash buffer (20 mM tris, pH 8.0, 300 mM KCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40) with protease inhibitor cocktail (Sigma) and proteins were boiled in Lammeli buffer. .. Samples were analysed by SDS–PAGE and western blotting probed with either Myc antibody (Cell Signaling, catalog #2276S) or BMI1 antibody (Millipore, catalog #05-637).

    Magnetic Beads:

    Article Title: Label-free electrical sensing of bacteria in eye wash samples: A step towards point-of-care detection of pathogens in patients with infectious keratitis
    Article Snippet: .. Streptavidin-coated magnetic beads (1 µm diameter, Thermo fisher scientific-Pierce™ Streptavidin Magnetic Beads 88816) were washed three times using PBS in a micro-centrifuge tube. .. All washing steps were done with the help of a magnetic stand (Millipore Magna GriP 20–400).

    Article Title: The estrogen receptor variants β2 and β5 induce stem cell characteristics and chemotherapy resistance in prostate cancer through activation of hypoxic signaling
    Article Snippet: .. 10 µl streptavidin magnetic beads (Pierce Rockford, IL) were washed in NETN buffer and incubated with 300 µl cellular extract for 2 hours in the cold room. .. Beads were washed (3 × 10 minutes rotation) with 300 μl NETN and boiled with 20 μl of 2× sample loading buffer [65.8 mM Tris HCl (pH 6.8), with 2.1% SDS, 26.3% (w/v) glycerol, and 0.01% bromophenol blue] and subjected to SDS-PAGE and transferred to nitrocellulose membrane for Western blot.

    Article Title: Generation and Nuclear Translocation of Sumoylated Transmembrane Fragment of Cell Adhesion Molecule L1
    Article Snippet: .. For isolation of biotinylated proteins after cell surface biotinylation, streptavidin-conjugated magnetic beads (Pierce) were incubated with cell lysates or cellular subfractions overnight at 4 °C. ..

    Article Title: BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization
    Article Snippet: .. Lysate was clarified by centrifugation and streptavidin magnetic beads (Pierce) were added to each sample and incubated at 4 °C with rotation for 16 h. Beads were washed 4 times with wash buffer (20 mM tris, pH 8.0, 300 mM KCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40) with protease inhibitor cocktail (Sigma) and proteins were boiled in Lammeli buffer. .. Samples were analysed by SDS–PAGE and western blotting probed with either Myc antibody (Cell Signaling, catalog #2276S) or BMI1 antibody (Millipore, catalog #05-637).

    Article Title: Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish
    Article Snippet: .. LPS pulldown assay Biotinylated-ultrapure LPS and Biotinylated-Pam3CSK4 from E. coli O111:B4 (InvivoGen) were incubated with Pierce™ Streptavidin Magnetic Beads (Thermo Scientific) for 6 h. The beads were then centrifuged at 800×g for 1 min and the supernatant was removed. .. One milliliter of purified HA-tagged caspy2, caspy2 (C296A), caspy2 (ΔPYD), caspy2 PYD, caspy, or caspy PYD protein in HEK293T cells was mixed with LPS or Pam3CSK4-bound beads at 4 °C overnight, after which, the beads were again centrifuged at 800×g for 1 min and the supernatant was removed.

    Article Title: Surface L-type Ca2+ channel expression levels are increased in aged hippocampus
    Article Snippet: .. Alternate slices from the dorsal half of both left and right hippocampi were exposed to 1 mg mL−1 Sulfo-NHS–SS–biot in-labeling reagent (Pierce) for 30 min before separating each hippocampal region for processing and isolation of surface proteins using streptavidin magnetic beads (Pierce). .. Immunoblotting with p1928, CNC1, and Ab144 antibody was performed after SDS-PAGE separation of total region lysates (input) and biotinylated (surface) fraction proteins.

    Incubation:

    Article Title: Carbonic anhydrase-related protein CA10 is an evolutionarily conserved pan-neurexin ligand
    Article Snippet: .. Lysates were cleared by centrifugation at 20,000 × g for 15 min, diluted 1:1 in RIPA, and, following removal of the input fraction, incubated with magnetic streptavidin beads (Pierce) under orbital rotation for 3 h at 4 °C. ..

    Article Title: The estrogen receptor variants β2 and β5 induce stem cell characteristics and chemotherapy resistance in prostate cancer through activation of hypoxic signaling
    Article Snippet: .. 10 µl streptavidin magnetic beads (Pierce Rockford, IL) were washed in NETN buffer and incubated with 300 µl cellular extract for 2 hours in the cold room. .. Beads were washed (3 × 10 minutes rotation) with 300 μl NETN and boiled with 20 μl of 2× sample loading buffer [65.8 mM Tris HCl (pH 6.8), with 2.1% SDS, 26.3% (w/v) glycerol, and 0.01% bromophenol blue] and subjected to SDS-PAGE and transferred to nitrocellulose membrane for Western blot.

    Article Title: Generation and Nuclear Translocation of Sumoylated Transmembrane Fragment of Cell Adhesion Molecule L1
    Article Snippet: .. For isolation of biotinylated proteins after cell surface biotinylation, streptavidin-conjugated magnetic beads (Pierce) were incubated with cell lysates or cellular subfractions overnight at 4 °C. ..

    Article Title: BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization
    Article Snippet: .. Lysate was clarified by centrifugation and streptavidin magnetic beads (Pierce) were added to each sample and incubated at 4 °C with rotation for 16 h. Beads were washed 4 times with wash buffer (20 mM tris, pH 8.0, 300 mM KCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40) with protease inhibitor cocktail (Sigma) and proteins were boiled in Lammeli buffer. .. Samples were analysed by SDS–PAGE and western blotting probed with either Myc antibody (Cell Signaling, catalog #2276S) or BMI1 antibody (Millipore, catalog #05-637).

    Article Title: Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish
    Article Snippet: .. LPS pulldown assay Biotinylated-ultrapure LPS and Biotinylated-Pam3CSK4 from E. coli O111:B4 (InvivoGen) were incubated with Pierce™ Streptavidin Magnetic Beads (Thermo Scientific) for 6 h. The beads were then centrifuged at 800×g for 1 min and the supernatant was removed. .. One milliliter of purified HA-tagged caspy2, caspy2 (C296A), caspy2 (ΔPYD), caspy2 PYD, caspy, or caspy PYD protein in HEK293T cells was mixed with LPS or Pam3CSK4-bound beads at 4 °C overnight, after which, the beads were again centrifuged at 800×g for 1 min and the supernatant was removed.

    Protease Inhibitor:

    Article Title: BMI1 regulates PRC1 architecture and activity through homo- and hetero-oligomerization
    Article Snippet: .. Lysate was clarified by centrifugation and streptavidin magnetic beads (Pierce) were added to each sample and incubated at 4 °C with rotation for 16 h. Beads were washed 4 times with wash buffer (20 mM tris, pH 8.0, 300 mM KCl, 1 mM EDTA, 10% glycerol, 0.1% NP-40) with protease inhibitor cocktail (Sigma) and proteins were boiled in Lammeli buffer. .. Samples were analysed by SDS–PAGE and western blotting probed with either Myc antibody (Cell Signaling, catalog #2276S) or BMI1 antibody (Millipore, catalog #05-637).

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    Thermo Fisher streptavidin coated magnetic beads
    Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on <t>streptavidin-beads</t> analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .
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    Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on streptavidin-beads analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .

    Journal: Molecular cell

    Article Title: Galectins control mTOR in response to endomembrane damage

    doi: 10.1016/j.molcel.2018.03.009

    Figure Lengend Snippet: Gal8 is in dynamic complexes with mTOR and its regulators and adaptors (A) Galectin puncta formation in response to GPN. Cells expressing YFP-galectin fusions were treated with 100 μM GPN or without (Ctrl) in full medium for 1 h and galectin puncta quantified by HC. Left, images of galectins 1, 3, 8, and 9. White masks, algorithm-defined cell boundaries (primary objects); green masks, computer-identified galectin puncta (target objects). (B) Co-immunoprecipitation (Co-IP) analysis of galectins and mTOR or RagA. Cells expressing FLAG-tagged galectins were subjected to anti-FLAG immunoprecipitation followed by immunoblotting for endogenous mTOR or RagA. (C) Co-IP analysis of endogenous proteins in macrophage-like cells treated with 100 μM GPN in full medium1 h. IP: anti-Gal8; immunoblotting: endogenous RagA, p14, mTOR and Raptor. (D) APEX2 proximity biotinylation analysis. Cells were transfected with APEX2 fusions with Gal3, 8 and 9, incubated or not with biotin-phenol, pulsed with H 2 O 2 , and biotinylated proteins affinity-isolated on streptavidin-beads analyzed by immunoblotting. (E) Proximity biotinylation as in D in response to GPN. BP, biotin-phenol. (F)(i-ii) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged p18 with GST, or GST-tagged Gal8 and Gal9. Data (% binding). (G)(i-ii) GST pulldown assay of in vitro translated Myc-tagged Gal8 or Gal9 with GST or GST-tagged RagB/D. Data as in F. (H) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 (negative control) or RagB variants (RagB WT , RagB T54L or RagB Q99L ) were subjected to anti-GFP IP, followed by immunoblotting for FLAG-tagged proteins or GFP. (I) Cells transfected with GFP-Gal8 and FLAG-tagged metap2 or RagC variants (RagC WT , RagC S75L or RagC Q120L .

    Article Snippet: Streptavidin–coated magnetic beads (ThermoFisher Scientific) were washed with RIPA lysis buffer.

    Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Incubation, Isolation, GST Pulldown Assay, In Vitro, Binding Assay, Negative Control

    3D Schematic of the presented mechanism for bacteria detection using electrical sensing of pathogen lysate Process flow for bacteria capture and detection: (i) Sample containing target pathogen is suspended with magnetic beads coated with streptavidin and conjugated with biotinylated antibodies and incubated for 30 minutes, (ii) The conjugated beads are isolated using a magnetic stand, (iii) The beads are washed using 10 % glycerol in DI water 4 times to remove unbound bacteria and electrically conductive solution. The captured bacteria are then lysed using 5 % Triton X-100 solution and sonication, (iv) Beads are isolated using magnetic stand, (v) The bacteria lysate is loaded onto a microchip with interdigitated electrodes for detection through impedance spectroscopy.

    Journal: Biosensors & bioelectronics

    Article Title: Label-free electrical sensing of bacteria in eye wash samples: A step towards point-of-care detection of pathogens in patients with infectious keratitis

    doi: 10.1016/j.bios.2016.12.035

    Figure Lengend Snippet: 3D Schematic of the presented mechanism for bacteria detection using electrical sensing of pathogen lysate Process flow for bacteria capture and detection: (i) Sample containing target pathogen is suspended with magnetic beads coated with streptavidin and conjugated with biotinylated antibodies and incubated for 30 minutes, (ii) The conjugated beads are isolated using a magnetic stand, (iii) The beads are washed using 10 % glycerol in DI water 4 times to remove unbound bacteria and electrically conductive solution. The captured bacteria are then lysed using 5 % Triton X-100 solution and sonication, (iv) Beads are isolated using magnetic stand, (v) The bacteria lysate is loaded onto a microchip with interdigitated electrodes for detection through impedance spectroscopy.

    Article Snippet: Streptavidin-coated magnetic beads (1 µm diameter, Thermo fisher scientific-Pierce™ Streptavidin Magnetic Beads 88816) were washed three times using PBS in a micro-centrifuge tube.

    Techniques: Flow Cytometry, Magnetic Beads, Incubation, Isolation, Sonication, MicroChIP Assay, Impedance Spectroscopy

    Biotinylated PNR502 localizes to aggregates in AM141 worms and is used to recover drug-adherent proteins. (A) Structure of biotinyl-PNR502. (B,C) AM141 adult worms were either untreated (B) or treated 26 h (C) with 10-μM PNR502 and then fed Alexa594-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were captured with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope. Q40::YFP is displayed as green, and Alexa594 as red fluorescence. Yellow fluorescence in (C) indicates the superposition of Q40::YFP with PNR502-Alexa594. (D) Caudal hippocampi, from normal age-matched controls (AMC) or AD patients (pools of three per group), were lysed and incubated 2 h at 4°C with 5-μM PNR502 or biotinyl-PNR502. M , size markers; lanes 1–4, proteins recovered from: 1 , unmodified PNR502; 2 , biotinyl-PNR502; 3 , equivalent portion of flow-through for unmodified PNR502; 4 , flow-through from biotinyl-PNR502.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: A Novel Microtubule-Binding Drug Attenuates and Reverses Protein Aggregation in Animal Models of Alzheimer’s Disease

    doi: 10.3389/fnmol.2019.00310

    Figure Lengend Snippet: Biotinylated PNR502 localizes to aggregates in AM141 worms and is used to recover drug-adherent proteins. (A) Structure of biotinyl-PNR502. (B,C) AM141 adult worms were either untreated (B) or treated 26 h (C) with 10-μM PNR502 and then fed Alexa594-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence images were captured with a Nikon DS-Fi2 camera mounted on a Nikon C2 inverted microscope. Q40::YFP is displayed as green, and Alexa594 as red fluorescence. Yellow fluorescence in (C) indicates the superposition of Q40::YFP with PNR502-Alexa594. (D) Caudal hippocampi, from normal age-matched controls (AMC) or AD patients (pools of three per group), were lysed and incubated 2 h at 4°C with 5-μM PNR502 or biotinyl-PNR502. M , size markers; lanes 1–4, proteins recovered from: 1 , unmodified PNR502; 2 , biotinyl-PNR502; 3 , equivalent portion of flow-through for unmodified PNR502; 4 , flow-through from biotinyl-PNR502.

    Article Snippet: Bound, lightly digested protein was recovered on streptavidin-coated magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA), and eluted peptides were analyzed by LC-MS/MS as described (Ayyadevara et al., , ).

    Techniques: Fluorescence, Inverted Microscopy, Incubation, Flow Cytometry