streptavidin magnetic beads  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Streptavidin Magnetic Beads
    Description:
    Streptavidin Magnetic Beads 5 ml
    Catalog Number:
    s1420s
    Price:
    318
    Size:
    5 ml
    Category:
    Magnetic Separation Equipment
    Buy from Supplier


    Structured Review

    New England Biolabs streptavidin magnetic beads
    Streptavidin Magnetic Beads
    Streptavidin Magnetic Beads 5 ml
    https://www.bioz.com/result/streptavidin magnetic beads/product/New England Biolabs
    Average 99 stars, based on 91 article reviews
    Price from $9.99 to $1999.99
    streptavidin magnetic beads - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Src kinase phosphorylates Notch1 to inhibit MAML binding"

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33920-y

    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.
    Figure Legend Snippet: Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Techniques Used: Purification, Western Blot, Expressing, Dominant Negative Mutation, Sequencing, Immunoprecipitation, Over Expression, Inhibition

    2) Product Images from "miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family"

    Article Title: miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family

    Journal: RNA Biology

    doi: 10.1080/15476286.2017.1422471

    RNA-Seq target validation. (A) qRT-PCR for nine candidate targets from RNA-Seq. Relative expression is significantly increased for LNA-106b compared to miR-106b in seven of the genes. There was a trend towards increased expression for LNA-106b compared to miR-106b for NCEH1 but it was not statistically significant (p = 0.07). FOS showed no significant change in expression by qRT-PCR. Dotted line represents expression level for scrambled control LNA. ITGA3 and HRAS are non-target negative control genes which show no significant expression change. Relative expression is given on the left y-axis and fold change is shown on the right y-axis. (B) Immunoblot showing transfection with miR-106b decreased protein levels of DR5, RB1, and E2F1 in Mz-ChA-1 cells. (C) Schematic of experimental design for capture of mRNA targets using biotinylated microRNA. Briefly, Mz-ChA-1 cells were transfected for 24 hours with either mature human miR-106b or C. elegans miR-67 which had been biotinylated. Cells were lysed and incubated with streptavidin-bound beads to capture biotinylated microRNA and associated mRNAs. Total RNA was isolated and relative expression of target mRNAs KLF2 and IL-8 was measured for enrichment by qRT-PCR. 18S was used as a control RNA. * p
    Figure Legend Snippet: RNA-Seq target validation. (A) qRT-PCR for nine candidate targets from RNA-Seq. Relative expression is significantly increased for LNA-106b compared to miR-106b in seven of the genes. There was a trend towards increased expression for LNA-106b compared to miR-106b for NCEH1 but it was not statistically significant (p = 0.07). FOS showed no significant change in expression by qRT-PCR. Dotted line represents expression level for scrambled control LNA. ITGA3 and HRAS are non-target negative control genes which show no significant expression change. Relative expression is given on the left y-axis and fold change is shown on the right y-axis. (B) Immunoblot showing transfection with miR-106b decreased protein levels of DR5, RB1, and E2F1 in Mz-ChA-1 cells. (C) Schematic of experimental design for capture of mRNA targets using biotinylated microRNA. Briefly, Mz-ChA-1 cells were transfected for 24 hours with either mature human miR-106b or C. elegans miR-67 which had been biotinylated. Cells were lysed and incubated with streptavidin-bound beads to capture biotinylated microRNA and associated mRNAs. Total RNA was isolated and relative expression of target mRNAs KLF2 and IL-8 was measured for enrichment by qRT-PCR. 18S was used as a control RNA. * p

    Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Negative Control, Transfection, Incubation, Isolation

    3) Product Images from "Src kinase phosphorylates Notch1 to inhibit MAML binding"

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33920-y

    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.
    Figure Legend Snippet: Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Techniques Used: Purification, Western Blot, Expressing, Dominant Negative Mutation, Sequencing, Immunoprecipitation, Over Expression, Inhibition

    4) Product Images from "Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions"

    Article Title: Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm588

    Single primer extension (SPEX) amplification. ( A ) Denaturation and hybridization of a single biotinylated primer to one target strand at the locus-of-interest. ( B ) Primer extension by a thermostable DNA polymerase until halted at the physical end of an aDNA template molecule; at a polymerase-blocking modified base [M]; or at an abasic site, some other non-coding lesion, or some kind of physical block [X]. Miscoding lesions [U] do not block primer extension, but result in altered sequences. Polymerases can catalyze the non-directed addition of a single 3′-terminal nucleotide [N] following primer extension to either the physical end of a fragmented aDNA template, or to an abasic site or other non-coding lesion. Single or multiple cycles of SPEX primer extension can be used. Biotinylated molecules were then bound to Streptavidin-coated beads and stringent washes removed everything else (e.g. aDNA template molecules, enzymes, buffer, etc). ( C ) Biotinylated primers and extended primers (with single-stranded, direct first-generation copies of individual aDNA template molecules) were then polyC-tailed using terminal transferase (TdT), followed again by bead-wash removal of TdT and buffer. ( D ) Locus-specific, primer-extended, polyC-tailed ssDNA molecules were then selectively amplified by PCR; using a partially-nested, locus-specific, SPEX-2 forward primer (Tables S1 and S2) and a polyG-based, 5′ adapter-tagged, reverse primer (Tables S1 and S2). ( E ) These products were amplified a final time by PCR using a further partially nested, locus-specific, SPEX-3 forward primer (Tables S1 and S2) and a 5′-adapter reverse primer (Tables S1 and S2). ( F ) The final products of SPEX amplification underwent restriction digestion, directional cloning and sequencing.
    Figure Legend Snippet: Single primer extension (SPEX) amplification. ( A ) Denaturation and hybridization of a single biotinylated primer to one target strand at the locus-of-interest. ( B ) Primer extension by a thermostable DNA polymerase until halted at the physical end of an aDNA template molecule; at a polymerase-blocking modified base [M]; or at an abasic site, some other non-coding lesion, or some kind of physical block [X]. Miscoding lesions [U] do not block primer extension, but result in altered sequences. Polymerases can catalyze the non-directed addition of a single 3′-terminal nucleotide [N] following primer extension to either the physical end of a fragmented aDNA template, or to an abasic site or other non-coding lesion. Single or multiple cycles of SPEX primer extension can be used. Biotinylated molecules were then bound to Streptavidin-coated beads and stringent washes removed everything else (e.g. aDNA template molecules, enzymes, buffer, etc). ( C ) Biotinylated primers and extended primers (with single-stranded, direct first-generation copies of individual aDNA template molecules) were then polyC-tailed using terminal transferase (TdT), followed again by bead-wash removal of TdT and buffer. ( D ) Locus-specific, primer-extended, polyC-tailed ssDNA molecules were then selectively amplified by PCR; using a partially-nested, locus-specific, SPEX-2 forward primer (Tables S1 and S2) and a polyG-based, 5′ adapter-tagged, reverse primer (Tables S1 and S2). ( E ) These products were amplified a final time by PCR using a further partially nested, locus-specific, SPEX-3 forward primer (Tables S1 and S2) and a 5′-adapter reverse primer (Tables S1 and S2). ( F ) The final products of SPEX amplification underwent restriction digestion, directional cloning and sequencing.

    Techniques Used: Amplification, Hybridization, Blocking Assay, Modification, Polymerase Chain Reaction, Clone Assay, Sequencing

    5) Product Images from "The Conserved C-Terminus of the PcrA/UvrD Helicase Interacts Directly with RNA Polymerase"

    Article Title: The Conserved C-Terminus of the PcrA/UvrD Helicase Interacts Directly with RNA Polymerase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078141

    Interaction between Escherichia coli UvrD helicase and RNAP. Pull down experiments were performed as described in the methods using the indicated biotinylated helicase as bait and E. coli cell extract as the prey. Proteins retained on streptavidin magnetic beads were compared against a mock control experiment in which the beads were not baited (upper panel). The samples were also analysed by western blot for the presence of RNAP using an antibody against the β subunit (lower panel). The positive control lane contains purified RNA polymerase.
    Figure Legend Snippet: Interaction between Escherichia coli UvrD helicase and RNAP. Pull down experiments were performed as described in the methods using the indicated biotinylated helicase as bait and E. coli cell extract as the prey. Proteins retained on streptavidin magnetic beads were compared against a mock control experiment in which the beads were not baited (upper panel). The samples were also analysed by western blot for the presence of RNAP using an antibody against the β subunit (lower panel). The positive control lane contains purified RNA polymerase.

    Techniques Used: Magnetic Beads, Western Blot, Positive Control, Purification

    Interactions between PcrA helicase and RNAP studied using magnetic bead pull down assays. (A) SDS-PAGE gel showing dose-dependent pull down of the core RNAP subunits from a DNA/RNA -depleted B. subtilis cell extract by biotinylated BsuPcrA (between 2 - 17 µg) on magnetic streptavidin beads. In the mock pull down, the beads were not baited with PcrA. (B) Depletion of nucleic acids from the cell extract by treatment with DNase and RNase has no apparent effect on the efficiency of RNAP pull down by BsuPcrA. (C) Interaction with Bacillus RNAP is specific to the PcrA helicase. The beads were baited with the helicase indicated and used to pull down proteins from B. subtilis cell extract (upper panel). The samples were analysed for the presence of RNA polymerase using an antibody against the β subunit (lower panel). The positive control lane contains purified RNA polymerase.
    Figure Legend Snippet: Interactions between PcrA helicase and RNAP studied using magnetic bead pull down assays. (A) SDS-PAGE gel showing dose-dependent pull down of the core RNAP subunits from a DNA/RNA -depleted B. subtilis cell extract by biotinylated BsuPcrA (between 2 - 17 µg) on magnetic streptavidin beads. In the mock pull down, the beads were not baited with PcrA. (B) Depletion of nucleic acids from the cell extract by treatment with DNase and RNase has no apparent effect on the efficiency of RNAP pull down by BsuPcrA. (C) Interaction with Bacillus RNAP is specific to the PcrA helicase. The beads were baited with the helicase indicated and used to pull down proteins from B. subtilis cell extract (upper panel). The samples were analysed for the presence of RNA polymerase using an antibody against the β subunit (lower panel). The positive control lane contains purified RNA polymerase.

    Techniques Used: SDS Page, Positive Control, Purification

    PcrA helicase interacts directly with purified RNA polymerase. Protein-protein interactions were monitored using surface plasmon resonance as described in the Methods section. (A) Example sensorgrams for the binding of BsuRNAP to immobilized GstPcrA. (B) Different biotinylated helicases were immobilized in a flow cell containing a streptavidin-coated sensor chip. Increasing concentrations of BsuRNAP were flowed over and binding was monitored as an increase in resonance units. The graph shows the collated results for binding of BsuRNAP to all immobilized helicases. C) his-tagged BsuRNAP was amine-coupled to a CM5 chip and the GstPcrA, EcUvrD and EcRep helicases were subsequently flowed over at different concentrations. Binding was monitored as an increase in resonance units.
    Figure Legend Snippet: PcrA helicase interacts directly with purified RNA polymerase. Protein-protein interactions were monitored using surface plasmon resonance as described in the Methods section. (A) Example sensorgrams for the binding of BsuRNAP to immobilized GstPcrA. (B) Different biotinylated helicases were immobilized in a flow cell containing a streptavidin-coated sensor chip. Increasing concentrations of BsuRNAP were flowed over and binding was monitored as an increase in resonance units. The graph shows the collated results for binding of BsuRNAP to all immobilized helicases. C) his-tagged BsuRNAP was amine-coupled to a CM5 chip and the GstPcrA, EcUvrD and EcRep helicases were subsequently flowed over at different concentrations. Binding was monitored as an increase in resonance units.

    Techniques Used: Purification, SPR Assay, Binding Assay, Flow Cytometry, Chromatin Immunoprecipitation

    6) Product Images from "Aberrant Glycosylation in the Human Trabecular Meshwork"

    Article Title: Aberrant Glycosylation in the Human Trabecular Meshwork

    Journal: Proteomics. Clinical applications

    doi: 10.1002/prca.201300031

    Glycosylation levels and protein glycosylation enzymatic activities in control and glaucomatous trabecular meshwork (TM). ( A ) Representative microscopic image of histochemical analyses (n=12 each glaucoma and control) of bound fluorescent-lectins in the TM. The cadaver TM from glaucomatous or control donors as indicated was probed using biotinylated lectins as indicated. The detection was based on Alexa ® -594 coupled streptavidin binding to biotinylated lectin. Images were taken on Leica TSP5 confocal microscope (Leica Corporation, Manheim, Germany) at 20x magnification. A tissue section that was not probed with any lectin served as negative control. ( B ) Representative Fluorophore-assisted carbohydrate electrophoresis (FACE ® ) analyses of 2-aminoacridone (AMAC) derivatized testicular hyaluronidase and chondroitinase ABC digested products from normal and glaucomatous TM as indicated. The disaccharide standards were obtained from Hyal-Dermato Disaccharide D-kit (cat# 400572-1; Seikagaku Corporation) To indicate equal loading, an identical aliquot was loaded on to a 10% SDS-PAGE and stained with Coomassie blue for protein staining with molecular markers as indicated presented in the bottom panel. ( C ) Relative quantification of glycosyltransferase immunoreactivities using ELISA and dotblot densitometric analyses as indicated. ( D ) Relative quantification of deglycosylase immunoreactivities using ELISA and dot blot densitometric analyses as indicated. The enzymatic immunoreactivity ratio (normalized to total protein/total of GAPDH) of indicated enzymes (C, D) with GAPDH was quantified using ELISA and dot blot analysis as indicated and as described in experimental procedures (n= 12 samples each for glaucoma and control). Results are mean ± standard deviation (*Significantly different results when compared between control and glaucoma donors by the two-tailed two sample t-test: *p
    Figure Legend Snippet: Glycosylation levels and protein glycosylation enzymatic activities in control and glaucomatous trabecular meshwork (TM). ( A ) Representative microscopic image of histochemical analyses (n=12 each glaucoma and control) of bound fluorescent-lectins in the TM. The cadaver TM from glaucomatous or control donors as indicated was probed using biotinylated lectins as indicated. The detection was based on Alexa ® -594 coupled streptavidin binding to biotinylated lectin. Images were taken on Leica TSP5 confocal microscope (Leica Corporation, Manheim, Germany) at 20x magnification. A tissue section that was not probed with any lectin served as negative control. ( B ) Representative Fluorophore-assisted carbohydrate electrophoresis (FACE ® ) analyses of 2-aminoacridone (AMAC) derivatized testicular hyaluronidase and chondroitinase ABC digested products from normal and glaucomatous TM as indicated. The disaccharide standards were obtained from Hyal-Dermato Disaccharide D-kit (cat# 400572-1; Seikagaku Corporation) To indicate equal loading, an identical aliquot was loaded on to a 10% SDS-PAGE and stained with Coomassie blue for protein staining with molecular markers as indicated presented in the bottom panel. ( C ) Relative quantification of glycosyltransferase immunoreactivities using ELISA and dotblot densitometric analyses as indicated. ( D ) Relative quantification of deglycosylase immunoreactivities using ELISA and dot blot densitometric analyses as indicated. The enzymatic immunoreactivity ratio (normalized to total protein/total of GAPDH) of indicated enzymes (C, D) with GAPDH was quantified using ELISA and dot blot analysis as indicated and as described in experimental procedures (n= 12 samples each for glaucoma and control). Results are mean ± standard deviation (*Significantly different results when compared between control and glaucoma donors by the two-tailed two sample t-test: *p

    Techniques Used: Binding Assay, Microscopy, Negative Control, Electrophoresis, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Dot Blot, Standard Deviation, Two Tailed Test

    7) Product Images from "Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments"

    Article Title: Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments

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

    doi: 10.1073/pnas.1208618109

    Slow dsRNA binding amplifies the length dependence of MDA5. ( A ) Analysis of the RNA-binding kinetics of MDA5 using biotin pull-down. As with the dissociation assays in , the level of MDA5 bound to dsRNA was monitored using streptavidin magnetic
    Figure Legend Snippet: Slow dsRNA binding amplifies the length dependence of MDA5. ( A ) Analysis of the RNA-binding kinetics of MDA5 using biotin pull-down. As with the dissociation assays in , the level of MDA5 bound to dsRNA was monitored using streptavidin magnetic

    Techniques Used: Binding Assay, RNA Binding Assay

    8) Product Images from "Covalent Surface Modification Effects on Single-Walled Carbon Nanotubes for Multimodal Optical Applications"

    Article Title: Covalent Surface Modification Effects on Single-Walled Carbon Nanotubes for Multimodal Optical Applications

    Journal: bioRxiv

    doi: 10.1101/837278

    Covalent modification of SWCNTs with amine-PEG2-biotin adds functional handles for avidin protein attachment. a) (GT) 15 -biotin-SWCNTs bind tetrameric avidin proteins such as neutravidin and streptavidin. b) A mass balance shows a higher percentage of bound (GT) 15 -biotin-SWCNTs on streptavidin beads compared to (GT) 15 -pristine-SWCNTs alone. c) AFM images show neutravidin protein bound to (GT) 15 -biotin-SWCNTs. d) (GT) 15 -biotin-SWCNTs immobilized on a neutravidin-coated microscopy surface (inset) and imaged with a near-infrared epifluorescence microscope with 721 nm excitation shows fluorescence response following exposure to 25 μM dopamine (addition denoted by the red arrow). The black line denotes the average fluorescence trace with individual microscopic regions of interest denoted by the gray lines
    Figure Legend Snippet: Covalent modification of SWCNTs with amine-PEG2-biotin adds functional handles for avidin protein attachment. a) (GT) 15 -biotin-SWCNTs bind tetrameric avidin proteins such as neutravidin and streptavidin. b) A mass balance shows a higher percentage of bound (GT) 15 -biotin-SWCNTs on streptavidin beads compared to (GT) 15 -pristine-SWCNTs alone. c) AFM images show neutravidin protein bound to (GT) 15 -biotin-SWCNTs. d) (GT) 15 -biotin-SWCNTs immobilized on a neutravidin-coated microscopy surface (inset) and imaged with a near-infrared epifluorescence microscope with 721 nm excitation shows fluorescence response following exposure to 25 μM dopamine (addition denoted by the red arrow). The black line denotes the average fluorescence trace with individual microscopic regions of interest denoted by the gray lines

    Techniques Used: Modification, Functional Assay, Avidin-Biotin Assay, Microscopy, Fluorescence

    9) Product Images from "Lactose repressor hinge domain independently binds DNA"

    Article Title: Lactose repressor hinge domain independently binds DNA

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.3372

    Purification and binding assays for His‐tagged LacI variants. (A) Purification of His 6 ‐LacI‐51 and His 6 ‐LacI‐59. Products of purification were examined using SDS‐PAGE to confirm purity (dashed boxes indicate samples used for binding assays). Lane 2 shows trypsin digestion products of wild‐type LacI (no His‐tag) for reference. (B) His‐tagged deletion mutants were used in a pull‐down assay with biotinylated O1 and O scram DNA bound to streptavidin‐coated beads with and without IPTG (I). Note that removal of the hinge helix in His 6 ‐LacI‐59 results in complete loss of DNA binding. (C) Electrophoretic mobility shift assays. Varying concentrations of His 6 ‐LacI‐51 and 10 −7 M P]‐labeled 40 bp O1 DNA (≤10 −11 M ) and equilibrated, followed by rapid loading onto a polyacrylamide gel. Note the loss of free DNA and detection of bound bands for His 6 ‐LacI‐51 at 10 −5 M protein; the lowest bound band exhibits slightly higher mobility compared to wild‐type LacI, as expected for this smaller tetramer. Note that wild‐type LacI was separated in this experiment from the His 6 ‐LacI‐51 and O1 DNA, and the dark line indicates removal of wells that contained other materials for analysis. (D) Band intensities for free DNA were derived from phosphorimaging of four separate experiments for His 6 ‐LacI‐51 and three separate experiments for wild‐type LacI; the values were normalized to the free O1 DNA band intensity for each experiment. Standard deviations for free DNA in samples with bound species are indicated.
    Figure Legend Snippet: Purification and binding assays for His‐tagged LacI variants. (A) Purification of His 6 ‐LacI‐51 and His 6 ‐LacI‐59. Products of purification were examined using SDS‐PAGE to confirm purity (dashed boxes indicate samples used for binding assays). Lane 2 shows trypsin digestion products of wild‐type LacI (no His‐tag) for reference. (B) His‐tagged deletion mutants were used in a pull‐down assay with biotinylated O1 and O scram DNA bound to streptavidin‐coated beads with and without IPTG (I). Note that removal of the hinge helix in His 6 ‐LacI‐59 results in complete loss of DNA binding. (C) Electrophoretic mobility shift assays. Varying concentrations of His 6 ‐LacI‐51 and 10 −7 M P]‐labeled 40 bp O1 DNA (≤10 −11 M ) and equilibrated, followed by rapid loading onto a polyacrylamide gel. Note the loss of free DNA and detection of bound bands for His 6 ‐LacI‐51 at 10 −5 M protein; the lowest bound band exhibits slightly higher mobility compared to wild‐type LacI, as expected for this smaller tetramer. Note that wild‐type LacI was separated in this experiment from the His 6 ‐LacI‐51 and O1 DNA, and the dark line indicates removal of wells that contained other materials for analysis. (D) Band intensities for free DNA were derived from phosphorimaging of four separate experiments for His 6 ‐LacI‐51 and three separate experiments for wild‐type LacI; the values were normalized to the free O1 DNA band intensity for each experiment. Standard deviations for free DNA in samples with bound species are indicated.

    Techniques Used: Purification, Binding Assay, SDS Page, Pull Down Assay, Electrophoretic Mobility Shift Assay, Labeling, Derivative Assay

    10) Product Images from "Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization"

    Article Title: Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

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

    doi: 10.1073/pnas.1524198113

    Ascorbate-dependent biotin switch assay after hind limb ischemia. The muscles were removed 3 d after hind limb ischemia surgery from WT and Glrx KO mice and processed for the ascorbate-dependent biotin switch assay for detection of S -nitrosylation. Biotin-labeled proteins were separated on SDS/PAGE and detected by dye-conjugated streptavidin. Nonischemic muscles (Non-Isc) and ischemic muscles (Isc) from each mouse are shown. Experiments were repeated three times with similar results.
    Figure Legend Snippet: Ascorbate-dependent biotin switch assay after hind limb ischemia. The muscles were removed 3 d after hind limb ischemia surgery from WT and Glrx KO mice and processed for the ascorbate-dependent biotin switch assay for detection of S -nitrosylation. Biotin-labeled proteins were separated on SDS/PAGE and detected by dye-conjugated streptavidin. Nonischemic muscles (Non-Isc) and ischemic muscles (Isc) from each mouse are shown. Experiments were repeated three times with similar results.

    Techniques Used: Biotin Switch Assay, Mouse Assay, Labeling, SDS Page

    Biotin switch assay of gastrocnemius muscles. The muscles were removed 3 d after hind limb ischemia surgery from WT and Glrx . Biotin-labeled proteins were separated on SDS/PAGE in a nonreducing condition and detected by dye-conjugated streptavidin. Nonischemic muscles (Non-Isc) and ischemic muscles (Isc) from each mouse are shown. Experiments were repeated three times with similar results.
    Figure Legend Snippet: Biotin switch assay of gastrocnemius muscles. The muscles were removed 3 d after hind limb ischemia surgery from WT and Glrx . Biotin-labeled proteins were separated on SDS/PAGE in a nonreducing condition and detected by dye-conjugated streptavidin. Nonischemic muscles (Non-Isc) and ischemic muscles (Isc) from each mouse are shown. Experiments were repeated three times with similar results.

    Techniques Used: Biotin Switch Assay, Labeling, SDS Page

    Effect of GSH adduct on HIF-1α stabilization. ( A ) Effect of GSSG-ethyl-ester on HIF-1α stabilization in C2C12 cells. After differentiation, C2C12 cells were treated with 50 μg GSSG-ethyl ester or PBS for 10 h. Representative Western blot of HIF-1α and β-tubulin ( Upper ) and densitometry analysis of HIF-1α normalized by β-tubulin ( Lower ) ( n = 8 each group). ( B ) Biotin switching assay for detection of HIF-1α reversible oxidative modification. DTT-dependent oxidative modified cysteines were labeled with biotin. Then biotin labeled protein was pull-downed using streptavidin beads. ( B ) Immunoblot of HIF-1α ( Upper Left ) and total reversible oxidative modified proteins detected by dye conjugated streptavidin ( Lower Left ) in a total sample. Immunoblot of HIF-1α ( Upper Right ) and total reversible oxidative modified proteins ( Lower Right ) in pulled down proteins. ( C ) Identification of Cys 520 GSH adduct by MS. GSH adduct of Cys 520 was detected from elastase fragment 520 CFYVDSDMV 528 . The actual mass of this fragment was 1,939.48 ( m/z = 700.25 2+ ), which was 321 Da more than the original MW. The MS/MS analysis showed that this fragment modified at Cys 520 by GSH adduct (+305 Da) and Met 527 by oxidation (+16 Da). ( D and E ) C520S mutation decreased 2-AAPA–dependent stabilization of HIF-1α. Plasmids that included WT and C520S mutant HIF-1α were transfected to COS7 cells in which endogenous HIF-1α was deleted by CRISPR/Cas9. These cells were treated with 20 μmol/L 2-AAPA for 3 h. ( D ) Representative Western blot of HIF-1α and β-tubulin. ( E ) Densitometry analysis, data were expressed as HIF-1α induction ratio of 2-AAPA–treated cells to respective vehicle-treated cells ( n = 4 each group). ( F ) Western blotting analysis following Co IP of HA-VHL overexpressed cell lysate and GSSG-treated Flag-tagged WT or C520S mutant HIF-1α–overexpressed cell lysate by anti-HA antibody. Detection of Flag-tagged HIF-1α and HA-tagged VHL was performed by anti-Flag antibody and anti-VHL antibody, respectively. Experiments were repeated three times with similar results. * P
    Figure Legend Snippet: Effect of GSH adduct on HIF-1α stabilization. ( A ) Effect of GSSG-ethyl-ester on HIF-1α stabilization in C2C12 cells. After differentiation, C2C12 cells were treated with 50 μg GSSG-ethyl ester or PBS for 10 h. Representative Western blot of HIF-1α and β-tubulin ( Upper ) and densitometry analysis of HIF-1α normalized by β-tubulin ( Lower ) ( n = 8 each group). ( B ) Biotin switching assay for detection of HIF-1α reversible oxidative modification. DTT-dependent oxidative modified cysteines were labeled with biotin. Then biotin labeled protein was pull-downed using streptavidin beads. ( B ) Immunoblot of HIF-1α ( Upper Left ) and total reversible oxidative modified proteins detected by dye conjugated streptavidin ( Lower Left ) in a total sample. Immunoblot of HIF-1α ( Upper Right ) and total reversible oxidative modified proteins ( Lower Right ) in pulled down proteins. ( C ) Identification of Cys 520 GSH adduct by MS. GSH adduct of Cys 520 was detected from elastase fragment 520 CFYVDSDMV 528 . The actual mass of this fragment was 1,939.48 ( m/z = 700.25 2+ ), which was 321 Da more than the original MW. The MS/MS analysis showed that this fragment modified at Cys 520 by GSH adduct (+305 Da) and Met 527 by oxidation (+16 Da). ( D and E ) C520S mutation decreased 2-AAPA–dependent stabilization of HIF-1α. Plasmids that included WT and C520S mutant HIF-1α were transfected to COS7 cells in which endogenous HIF-1α was deleted by CRISPR/Cas9. These cells were treated with 20 μmol/L 2-AAPA for 3 h. ( D ) Representative Western blot of HIF-1α and β-tubulin. ( E ) Densitometry analysis, data were expressed as HIF-1α induction ratio of 2-AAPA–treated cells to respective vehicle-treated cells ( n = 4 each group). ( F ) Western blotting analysis following Co IP of HA-VHL overexpressed cell lysate and GSSG-treated Flag-tagged WT or C520S mutant HIF-1α–overexpressed cell lysate by anti-HA antibody. Detection of Flag-tagged HIF-1α and HA-tagged VHL was performed by anti-Flag antibody and anti-VHL antibody, respectively. Experiments were repeated three times with similar results. * P

    Techniques Used: Western Blot, Modification, Labeling, Mass Spectrometry, Mutagenesis, Transfection, CRISPR, Co-Immunoprecipitation Assay

    Biotin switch assay of the Glrx knockdown cell. Glrx knockdown in the C2C12 cell was performed by siGlrx. After C2C12 cells were differentiated, these cells were lysed, and the biotin switch assay was performed. Experiments were repeated three times with similar results. Biotin-labeled proteins were separated on SDS/PAGE in a nonreducing condition and detected by dye-conjugated streptavidin or anti–HIF-1α antibody.
    Figure Legend Snippet: Biotin switch assay of the Glrx knockdown cell. Glrx knockdown in the C2C12 cell was performed by siGlrx. After C2C12 cells were differentiated, these cells were lysed, and the biotin switch assay was performed. Experiments were repeated three times with similar results. Biotin-labeled proteins were separated on SDS/PAGE in a nonreducing condition and detected by dye-conjugated streptavidin or anti–HIF-1α antibody.

    Techniques Used: Biotin Switch Assay, Labeling, SDS Page

    11) Product Images from "Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System"

    Article Title: Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

    Journal: Molecules

    doi: 10.3390/molecules17032855

    Sequence analysis of the 48-mer transcripts containing Biotin- Pa or U at position 21. The 5′ 3 2 P-labeled transcripts were partially digested with either RNase T1 (T1) or with alkali (AL). A portion of the partially alkali-digested transcripts was treated with streptavidin magnetic beads, to capture the RNA fragments containing Biotin- Pa (AL+SA). Each digested fragment was analyzed on a 10% polyacrylamide gel containing 7 M urea.
    Figure Legend Snippet: Sequence analysis of the 48-mer transcripts containing Biotin- Pa or U at position 21. The 5′ 3 2 P-labeled transcripts were partially digested with either RNase T1 (T1) or with alkali (AL). A portion of the partially alkali-digested transcripts was treated with streptavidin magnetic beads, to capture the RNA fragments containing Biotin- Pa (AL+SA). Each digested fragment was analyzed on a 10% polyacrylamide gel containing 7 M urea.

    Techniques Used: Sequencing, Labeling, Magnetic Beads

    12) Product Images from "Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity"

    Article Title: Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity

    Journal: RNA

    doi: 10.1261/rna.2701111

    Pre-miR-886 is physically associated with PKR and suppresses it from activation. ( A ) Western blotting of PKR ( top panel) in the 0.6 M KCl fraction eluted from the indicated biotinylated RNA–streptavidin bead complex (see Materials and Methods).
    Figure Legend Snippet: Pre-miR-886 is physically associated with PKR and suppresses it from activation. ( A ) Western blotting of PKR ( top panel) in the 0.6 M KCl fraction eluted from the indicated biotinylated RNA–streptavidin bead complex (see Materials and Methods).

    Techniques Used: Activation Assay, Western Blot

    13) Product Images from "Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB"

    Article Title: Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146064

    Enrichment workflow using HpaII. (A) Biotinylated HpaII enzyme is conjugated to streptavidin coated magnetic beads. A DNA mixture can then be added to the conjugated beads and following incubation the mixture is segregated into fractions that are bound (containing majority of Y . pestis ) or unbound (containing majority of human) to the beads. (B) Adding salt to the binding buffer enhances segregation of human (blue bars) from Y . pestis (green bars).
    Figure Legend Snippet: Enrichment workflow using HpaII. (A) Biotinylated HpaII enzyme is conjugated to streptavidin coated magnetic beads. A DNA mixture can then be added to the conjugated beads and following incubation the mixture is segregated into fractions that are bound (containing majority of Y . pestis ) or unbound (containing majority of human) to the beads. (B) Adding salt to the binding buffer enhances segregation of human (blue bars) from Y . pestis (green bars).

    Techniques Used: Magnetic Beads, Incubation, Binding Assay

    14) Product Images from "An RNA polymerase ribozyme that synthesizes its own ancestor"

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor

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

    doi: 10.1073/pnas.1914282117

    In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.
    Figure Legend Snippet: In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.

    Techniques Used: In Vitro, Amplification, Functional Assay, Hybridization, Magnetic Beads, Polymerase Chain Reaction, Sequencing, Labeling

    15) Product Images from "SPACE exploration of chromatin proteome to reveal associated RNA-binding proteins"

    Article Title: SPACE exploration of chromatin proteome to reveal associated RNA-binding proteins

    Journal: bioRxiv

    doi: 10.1101/2020.07.13.200212

    Overview of the SPACE and SPACE-SICAP procedures. 1: Cells are crosslinked by 1% formaldehyde, resuspended in the lysis buffer containing guanidinium, and iso-propanol and silica magnetic beads are added to the lysate. 2: Chromatin binds to the magnetic beads, and is separated from the lysate. 3: The beads are washed with lysis buffer and ethanol, and are treated with RNase A. 4a: In SPACE, the beads are washed again with ethanol and Acetonitrile, and Trypsin/LysC are added to digest the chromatin-associated proteins on the beads. 4b: In SPACE-SICAP, chromatin is eluted from the silica magnetic beads DNA is end-labelled by TdT and biotin-dCTP, andt chromatin is captured by protease-resistant streptavidin beads. 5: Chromatin is stringently washed, and chromatin-associated proteins are digested by LysC. 6: The supernatant is collected, and digested with Trypsin to generate peptides for chromatin and DNA-binders (SPACE-SICAP I). The streptavidin beads are washed and heated to reverse the crosslinking, and the released peptides are digested by Trypsin to generate peptides of DNA-binders (SPACE-SICAP II,). The peptides are cleaned and injected to the mass spectrometer.
    Figure Legend Snippet: Overview of the SPACE and SPACE-SICAP procedures. 1: Cells are crosslinked by 1% formaldehyde, resuspended in the lysis buffer containing guanidinium, and iso-propanol and silica magnetic beads are added to the lysate. 2: Chromatin binds to the magnetic beads, and is separated from the lysate. 3: The beads are washed with lysis buffer and ethanol, and are treated with RNase A. 4a: In SPACE, the beads are washed again with ethanol and Acetonitrile, and Trypsin/LysC are added to digest the chromatin-associated proteins on the beads. 4b: In SPACE-SICAP, chromatin is eluted from the silica magnetic beads DNA is end-labelled by TdT and biotin-dCTP, andt chromatin is captured by protease-resistant streptavidin beads. 5: Chromatin is stringently washed, and chromatin-associated proteins are digested by LysC. 6: The supernatant is collected, and digested with Trypsin to generate peptides for chromatin and DNA-binders (SPACE-SICAP I). The streptavidin beads are washed and heated to reverse the crosslinking, and the released peptides are digested by Trypsin to generate peptides of DNA-binders (SPACE-SICAP II,). The peptides are cleaned and injected to the mass spectrometer.

    Techniques Used: Lysis, Magnetic Beads, Injection, Mass Spectrometry

    16) Product Images from "3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids"

    Article Title: 3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

    Journal: Journal of structural biology

    doi: 10.1016/j.jsb.2019.06.004

    Processing of the streptavidin lattice in our images of Pol II EC(CPD) on streptavidin affinity support grids. (A) A representative micrograph showing Pol II EC(CPD) particles on a streptavidin crystal lattice. (B) FFT of the micrograph in (A) showing diffraction spots from the streptavidin crystal lattice. (C) ). Each diffraction spot is shown as a square and the size of the square indicates the signal to noise ratio for that spot. (D) The same micrograph shown in (A) after removing the streptavidin lattice using Fourier filtering. (E) FFT of the micrograph in (D) showing the disappearance of the diffraction spots. Zoom-ins of the regions highlighted in (A) and (D) (black squares), and (B) and (E) (white square) are shown below (for A and B) or above (for D and E) the corresponding panels. (F) Plot of the resolution at which the cross-correlation between the calculated and observed CTFs is 0.5 as a function of the estimated resolution of the streptavidin crystal. The grey horizontal line shows the CTF resolution cutoff used for image processing (4.5Å). All the micrographs above the grey line were discarded. The grey (no cutoff), orange (4.5Å cutoff) and red (2.9 Å cutoff) vertical dashed lines show the diffraction spot resolution cutoffs used for different reconstruction trials. Micrographs to the left of the dashed lines (shown by horizontal lines of the corresponding colors) were used for the reconstructions. The resolutions of the maps from these trials along with the number of particles contributing to the reconstructions are shown above the graph. The map reported in this work was the one obtained without imposing a diffraction resolution cutoff.
    Figure Legend Snippet: Processing of the streptavidin lattice in our images of Pol II EC(CPD) on streptavidin affinity support grids. (A) A representative micrograph showing Pol II EC(CPD) particles on a streptavidin crystal lattice. (B) FFT of the micrograph in (A) showing diffraction spots from the streptavidin crystal lattice. (C) ). Each diffraction spot is shown as a square and the size of the square indicates the signal to noise ratio for that spot. (D) The same micrograph shown in (A) after removing the streptavidin lattice using Fourier filtering. (E) FFT of the micrograph in (D) showing the disappearance of the diffraction spots. Zoom-ins of the regions highlighted in (A) and (D) (black squares), and (B) and (E) (white square) are shown below (for A and B) or above (for D and E) the corresponding panels. (F) Plot of the resolution at which the cross-correlation between the calculated and observed CTFs is 0.5 as a function of the estimated resolution of the streptavidin crystal. The grey horizontal line shows the CTF resolution cutoff used for image processing (4.5Å). All the micrographs above the grey line were discarded. The grey (no cutoff), orange (4.5Å cutoff) and red (2.9 Å cutoff) vertical dashed lines show the diffraction spot resolution cutoffs used for different reconstruction trials. Micrographs to the left of the dashed lines (shown by horizontal lines of the corresponding colors) were used for the reconstructions. The resolutions of the maps from these trials along with the number of particles contributing to the reconstructions are shown above the graph. The map reported in this work was the one obtained without imposing a diffraction resolution cutoff.

    Techniques Used:

    Biotinylation of an RNA Polymerase II Elongation Complex stalled at a cyclobutane pyrimidine dimer (CPD) lesion (Pol II EC(CPD)). (A) Schematic diagram of the assay used to determine the degree of biotinylation of Pol II EC(CPD). (B) ). (C) Negative stain EM micrographs of biotinylated Pol II EC(CPD) on continuous carbon (top) and streptavidin monolayer (bottom) grids.
    Figure Legend Snippet: Biotinylation of an RNA Polymerase II Elongation Complex stalled at a cyclobutane pyrimidine dimer (CPD) lesion (Pol II EC(CPD)). (A) Schematic diagram of the assay used to determine the degree of biotinylation of Pol II EC(CPD). (B) ). (C) Negative stain EM micrographs of biotinylated Pol II EC(CPD) on continuous carbon (top) and streptavidin monolayer (bottom) grids.

    Techniques Used: Staining

    3.1Å cryo-EM map of a randomly biotinylated Pol II EC(CPD) obtained using monolayer streptavidin crystal affinity grids. (A) Locally-filtered Cryo-EM map of Pol II EC(CPD). The map is segmented into the following regions: Pol II (grey), template strand (TS) (blue), non-template strand (NTS) (green) and RNA strand (red). The inset highlights two domains of Pol II we refer to in the text on a cartoon representation of the structure. (B–D) Representative regions of the Pol II EC(CPD) map: an alpha helix (B), a beta sheet (C), and Watson-Crick base pairing between the TS and the nascent RNA (D). The map is shown in transparent grey with the atomic model fitted into the density. The color code for the atomic model is the same as in A. (E) Pol II EC(CPD) map colored according to local resolution. The top panel shows the surface views of the map in different orientations and the bottom panel shows the corresponding cross-sections to highlight the resolutions in the interior of the map. (F) Schematic representations of the expected (top) and observed (bottom) positions of the bases in the transcription scaffold. The grey cylinder represents the bridge helix. For the observed (bottom) scaffold the dash indicates that the CPD lesion (orange) is connected to the TS by a phosphodiester bond. Bases that are disordered in our map are shown in lighter color. The 5’ and 3’ ends of the TS (blue) and NTS (green) are indicated. (G) Top view of the atomic model of the transcription scaffold. The bridge helix is shown for reference. The inset shows the CPD lesion fitted to the segmented TS density. αβ
    Figure Legend Snippet: 3.1Å cryo-EM map of a randomly biotinylated Pol II EC(CPD) obtained using monolayer streptavidin crystal affinity grids. (A) Locally-filtered Cryo-EM map of Pol II EC(CPD). The map is segmented into the following regions: Pol II (grey), template strand (TS) (blue), non-template strand (NTS) (green) and RNA strand (red). The inset highlights two domains of Pol II we refer to in the text on a cartoon representation of the structure. (B–D) Representative regions of the Pol II EC(CPD) map: an alpha helix (B), a beta sheet (C), and Watson-Crick base pairing between the TS and the nascent RNA (D). The map is shown in transparent grey with the atomic model fitted into the density. The color code for the atomic model is the same as in A. (E) Pol II EC(CPD) map colored according to local resolution. The top panel shows the surface views of the map in different orientations and the bottom panel shows the corresponding cross-sections to highlight the resolutions in the interior of the map. (F) Schematic representations of the expected (top) and observed (bottom) positions of the bases in the transcription scaffold. The grey cylinder represents the bridge helix. For the observed (bottom) scaffold the dash indicates that the CPD lesion (orange) is connected to the TS by a phosphodiester bond. Bases that are disordered in our map are shown in lighter color. The 5’ and 3’ ends of the TS (blue) and NTS (green) are indicated. (G) Top view of the atomic model of the transcription scaffold. The bridge helix is shown for reference. The inset shows the CPD lesion fitted to the segmented TS density. αβ

    Techniques Used:

    The accessibility of surface-exposed lysines correlates with particle orientation. (A) 3D FSC plot for the Pol II EC(CPD) reconstruction. (B) Euler angle distribution of the particles contributing to the reconstruction. (C–E) Solvent exposed surface area (SASA) of the lysine primary amines available for biotinylation. We used the atomic model of a previously published Pol II elongation complex (PDB ID: 1Y77) to represent the Pol II EC(CPD) in these panels. (C) A rare (top) and a common (bottom) view of Pol II EC(CPD) are shown along with the corresponding plots of Euler angle distribution. (D) This panel shows the surfaces of Pol II that must face the SA layer to lead to the views shown in (C). The primary amines of lysines are color-coded according to their solvent accessible surface area (SASA), with the least exposed amines shown in yellow and the most exposed ones in red. (E) This panel shows the same views shown in (D) but with a cutting plane adjusted such that only lysines whose biotin moieties would be capable of interacting with the streptavidin monolayer are shown. The cutting plane was set to ~29Å from the lysine’s amine, which is the length of a fully stretched biotinylation reagent molecule. The orientations and the cutting planes of the Pol II EC with respect the streptavidin monolayer (ochre rectangle) are shown next to each of the views in (D) and (E).
    Figure Legend Snippet: The accessibility of surface-exposed lysines correlates with particle orientation. (A) 3D FSC plot for the Pol II EC(CPD) reconstruction. (B) Euler angle distribution of the particles contributing to the reconstruction. (C–E) Solvent exposed surface area (SASA) of the lysine primary amines available for biotinylation. We used the atomic model of a previously published Pol II elongation complex (PDB ID: 1Y77) to represent the Pol II EC(CPD) in these panels. (C) A rare (top) and a common (bottom) view of Pol II EC(CPD) are shown along with the corresponding plots of Euler angle distribution. (D) This panel shows the surfaces of Pol II that must face the SA layer to lead to the views shown in (C). The primary amines of lysines are color-coded according to their solvent accessible surface area (SASA), with the least exposed amines shown in yellow and the most exposed ones in red. (E) This panel shows the same views shown in (D) but with a cutting plane adjusted such that only lysines whose biotin moieties would be capable of interacting with the streptavidin monolayer are shown. The cutting plane was set to ~29Å from the lysine’s amine, which is the length of a fully stretched biotinylation reagent molecule. The orientations and the cutting planes of the Pol II EC with respect the streptavidin monolayer (ochre rectangle) are shown next to each of the views in (D) and (E).

    Techniques Used:

    17) Product Images from "Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5–dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics"

    Article Title: Novel regulatory roles of Mff and Drp1 in E3 ubiquitin ligase MARCH5–dependent degradation of MiD49 and Mcl1 and control of mitochondrial dynamics

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-04-0208

    Identification of MARCH5-interacting proteins. (A) Cells were transfected with MARCH5(HA)BirA* along with biotin treatment (final concentration 50 mM). At ∼24 h after transfection, cells were fixed and stained for protein biotinylation (with Alexa 488–streptavidin; green on overlay image), MARCH5(HA)BirA* (with anti-HA mAb; blue on overlay image), and mitochondria (with anti-Tom20 polyclonal antibody; red on overlay image). MARCH5(HA)BirA*-expressing cells are overlaid in yellow. Asterisk indicates nontransfected cells. (B) Total cell lysates (INPUT) and streptavidin-purified biotinylated proteins (streptavidin pull down) obtained from cells transfected with the indicated constructs and treated with biotin as described in A. Approximately10% of inputs and streptavidin bead–purified biotinylated proteins (red rectangle in B) were used for SDS–PAGE. Biotinylated proteins were detected with HRP-streptavidin. (C) MARCH5-nteracting proteins (MARCH5 BioID assay summary; for details, see the text). (D) Cell lysates obtained from control-, MYC-MARCH5–, and MYC-MARCH5 H43W –transfected wild-type and Mff −/− HCT116 cells were subjected to MYC immunoprecipitation. Immunoprecipitated samples (left) and inputs (right) were analyzed by Western blot as indicated.
    Figure Legend Snippet: Identification of MARCH5-interacting proteins. (A) Cells were transfected with MARCH5(HA)BirA* along with biotin treatment (final concentration 50 mM). At ∼24 h after transfection, cells were fixed and stained for protein biotinylation (with Alexa 488–streptavidin; green on overlay image), MARCH5(HA)BirA* (with anti-HA mAb; blue on overlay image), and mitochondria (with anti-Tom20 polyclonal antibody; red on overlay image). MARCH5(HA)BirA*-expressing cells are overlaid in yellow. Asterisk indicates nontransfected cells. (B) Total cell lysates (INPUT) and streptavidin-purified biotinylated proteins (streptavidin pull down) obtained from cells transfected with the indicated constructs and treated with biotin as described in A. Approximately10% of inputs and streptavidin bead–purified biotinylated proteins (red rectangle in B) were used for SDS–PAGE. Biotinylated proteins were detected with HRP-streptavidin. (C) MARCH5-nteracting proteins (MARCH5 BioID assay summary; for details, see the text). (D) Cell lysates obtained from control-, MYC-MARCH5–, and MYC-MARCH5 H43W –transfected wild-type and Mff −/− HCT116 cells were subjected to MYC immunoprecipitation. Immunoprecipitated samples (left) and inputs (right) were analyzed by Western blot as indicated.

    Techniques Used: Transfection, Concentration Assay, Staining, Expressing, Purification, Construct, SDS Page, Immunoprecipitation, Western Blot

    18) Product Images from "“Hit-and-Run” transcription: de novo transcription initiated by a transient bZIP1 “hit” persists after the “run”"

    Article Title: “Hit-and-Run” transcription: de novo transcription initiated by a transient bZIP1 “hit” persists after the “run”

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2410-2

    TARGET - tU identifies actively transcribed TF targets. Schematic of the TARGET - tU system. a Protoplasts (plant cells dissociated from whole roots) transfected with a 35S::GR::TF construct are sequentially treated with: i) the nitrogen (N) signal transduced by the TF, ii) cycloheximide (CHX) to block translation, allowing RNA synthesis of only primary TF targets, iii) dexamethasone (DEX) to release the GR-TF fusion from the cytoplasmic heat shock complex (HSP), inducing nuclear import. Five hours after DEX-induction of TF nuclear localization, cells were exposed to iv) 4-thiouracil (4tU) so that thio-labeled UTP nucleotides are incorporated into newly synthesized RNA (see also c and Additional file 2: Figure S1). b Thiol-specific biotinylation and pull-down with streptavidin-coated magnetic beads enable selection of newly synthesized transcripts apart from pre-existing transcripts. c Timeline of the sequential treatments described in this study. Cell protoplasts were exposed to 4tU nucleobase 5 h after bZIP1 nuclear activation, to show the continued transcription of “hit-and run” targets
    Figure Legend Snippet: TARGET - tU identifies actively transcribed TF targets. Schematic of the TARGET - tU system. a Protoplasts (plant cells dissociated from whole roots) transfected with a 35S::GR::TF construct are sequentially treated with: i) the nitrogen (N) signal transduced by the TF, ii) cycloheximide (CHX) to block translation, allowing RNA synthesis of only primary TF targets, iii) dexamethasone (DEX) to release the GR-TF fusion from the cytoplasmic heat shock complex (HSP), inducing nuclear import. Five hours after DEX-induction of TF nuclear localization, cells were exposed to iv) 4-thiouracil (4tU) so that thio-labeled UTP nucleotides are incorporated into newly synthesized RNA (see also c and Additional file 2: Figure S1). b Thiol-specific biotinylation and pull-down with streptavidin-coated magnetic beads enable selection of newly synthesized transcripts apart from pre-existing transcripts. c Timeline of the sequential treatments described in this study. Cell protoplasts were exposed to 4tU nucleobase 5 h after bZIP1 nuclear activation, to show the continued transcription of “hit-and run” targets

    Techniques Used: Transfection, Construct, Blocking Assay, Labeling, Synthesized, Magnetic Beads, Selection, Activation Assay

    19) Product Images from "Separation of young and mature thrombocytes by a novel immuno-selection method"

    Article Title: Separation of young and mature thrombocytes by a novel immuno-selection method

    Journal: Blood cells, molecules & diseases

    doi: 10.1016/j.bcmd.2011.12.006

    Principle of thrombocyte isolation using magnetic beads. Y, young thrombocyte; M, mature thrombocyte; R, red cell; N, neutrophil; B, biotin labeled antibody; S, streptavidin beads attached to magnet. IgG/Cy3 and IgG/GPIIb are represented as Y shaped structures. Thrombocyte and neutrophil are shown in shaded circles. The triangular and square structures on the shaded circles represent Cy3 and GPIIb respectively. Red cells are shown in shaded ovals.
    Figure Legend Snippet: Principle of thrombocyte isolation using magnetic beads. Y, young thrombocyte; M, mature thrombocyte; R, red cell; N, neutrophil; B, biotin labeled antibody; S, streptavidin beads attached to magnet. IgG/Cy3 and IgG/GPIIb are represented as Y shaped structures. Thrombocyte and neutrophil are shown in shaded circles. The triangular and square structures on the shaded circles represent Cy3 and GPIIb respectively. Red cells are shown in shaded ovals.

    Techniques Used: Isolation, Magnetic Beads, Labeling

    Immunofluorescence images of blood cells. A, images showing blood cells incubated with anti-Cy3 antibody kept under a cover slip. Left panel, bright field image; middle panel, fluorescence image of the DiI labeled thrombocyte; right panel, fluorescence image of the streptavidin FITC labeled thrombocyte. B, images showing blood smears probed with biotinylated anti-GPIIb antibody followed by streptavidin FITC. Left panel, bright field image; right panel, fluorescence image of the streptavidin FITC labeled thrombocyte. Arrows indicate the thrombocytes.
    Figure Legend Snippet: Immunofluorescence images of blood cells. A, images showing blood cells incubated with anti-Cy3 antibody kept under a cover slip. Left panel, bright field image; middle panel, fluorescence image of the DiI labeled thrombocyte; right panel, fluorescence image of the streptavidin FITC labeled thrombocyte. B, images showing blood smears probed with biotinylated anti-GPIIb antibody followed by streptavidin FITC. Left panel, bright field image; right panel, fluorescence image of the streptavidin FITC labeled thrombocyte. Arrows indicate the thrombocytes.

    Techniques Used: Immunofluorescence, Incubation, Fluorescence, Labeling

    Slide showing streptavidin magnetic beads after pulling the DiI labeled young thrombocytes. A, bright field images and B, fluorescence images.
    Figure Legend Snippet: Slide showing streptavidin magnetic beads after pulling the DiI labeled young thrombocytes. A, bright field images and B, fluorescence images.

    Techniques Used: Magnetic Beads, Labeling, Fluorescence

    20) Product Images from "Determination of in vivo RNA kinetics using RATE-seq"

    Article Title: Determination of in vivo RNA kinetics using RATE-seq

    Journal: RNA

    doi: 10.1261/rna.045104.114

    RATE-seq enables in vivo measurement of RNA kinetics. ( A ) Overview of approach to equilibrium labeling and analysis using RATE-seq. The increase in labeled transcript with time Y ( t ) modeled using the relationship , where Y eq is the abundance of labeled transcript at steady state, α RNA is the transcript's degradation rate constant, α growth is the growth rate constant of the culture, t is the time after addition of label, and t d is a time delay between the addition of label and the time at which labeled transcripts can be detected. Red arrows indicate points at which the RNA samples are recovered following addition of 4tU. ( B ) Incorporation of 4tU conforms to approach to equilbrium kinetics. An equivalent quantity of biotinylated polyadenylated RNA from timepoints following addition of 4tU was bound to a membrane and visualized using streptavidin alkaline phosphatase and chemifluorescence. Values are shown along with the model fit.
    Figure Legend Snippet: RATE-seq enables in vivo measurement of RNA kinetics. ( A ) Overview of approach to equilibrium labeling and analysis using RATE-seq. The increase in labeled transcript with time Y ( t ) modeled using the relationship , where Y eq is the abundance of labeled transcript at steady state, α RNA is the transcript's degradation rate constant, α growth is the growth rate constant of the culture, t is the time after addition of label, and t d is a time delay between the addition of label and the time at which labeled transcripts can be detected. Red arrows indicate points at which the RNA samples are recovered following addition of 4tU. ( B ) Incorporation of 4tU conforms to approach to equilbrium kinetics. An equivalent quantity of biotinylated polyadenylated RNA from timepoints following addition of 4tU was bound to a membrane and visualized using streptavidin alkaline phosphatase and chemifluorescence. Values are shown along with the model fit.

    Techniques Used: In Vivo, Labeling

    21) Product Images from "A HSP60-targeting peptide for cell apoptosis imaging"

    Article Title: A HSP60-targeting peptide for cell apoptosis imaging

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2016.14

    Identification of HSP60 as the target of P17. ( a ) Protocols used for pull-down-assay-based analysis of binder of P17. ( b ) Biotin-P17 labeled streptavidin magnetic beads were used for pull-down assay from TRAIL-treated Jurkat cell extracts. Bound proteins were separated by SDS–PAGE. Absent from TRAIL-untreated and bead-control samples, a major band at 58 kDa was excised for mass spectrometry and identified as HSP60. ( c ) Mass spectrometry analysis identified peptides in HSP60 with 37% sequence coverage. Matched amino acids were shown in red. ( d ) Colocalization of FITC-P17 and anti-HSP60 (Alexa Fluor 594-conjugated) in TRAIL-treated Jurkat cells (Scale bars: 10 μm) observed by confocal microscopy (upper) and analyzed by Image-Pro Plus (bottom). ( e ) The schematic illustration for pull-down and western blot analysis. ( f ) Detection of HSP60 in pull-down samples by western blot and coomassie brilliant blue staining.
    Figure Legend Snippet: Identification of HSP60 as the target of P17. ( a ) Protocols used for pull-down-assay-based analysis of binder of P17. ( b ) Biotin-P17 labeled streptavidin magnetic beads were used for pull-down assay from TRAIL-treated Jurkat cell extracts. Bound proteins were separated by SDS–PAGE. Absent from TRAIL-untreated and bead-control samples, a major band at 58 kDa was excised for mass spectrometry and identified as HSP60. ( c ) Mass spectrometry analysis identified peptides in HSP60 with 37% sequence coverage. Matched amino acids were shown in red. ( d ) Colocalization of FITC-P17 and anti-HSP60 (Alexa Fluor 594-conjugated) in TRAIL-treated Jurkat cells (Scale bars: 10 μm) observed by confocal microscopy (upper) and analyzed by Image-Pro Plus (bottom). ( e ) The schematic illustration for pull-down and western blot analysis. ( f ) Detection of HSP60 in pull-down samples by western blot and coomassie brilliant blue staining.

    Techniques Used: Pull Down Assay, Labeling, Magnetic Beads, SDS Page, Mass Spectrometry, Sequencing, Confocal Microscopy, Western Blot, Staining

    22) Product Images from "TurboID-mediated proximity labelling of cytoophidium proteome in Drosophila"

    Article Title: TurboID-mediated proximity labelling of cytoophidium proteome in Drosophila

    Journal: bioRxiv

    doi: 10.1101/848283

    TurboID-mediated proximal biotinylation of CTPS cytoophidium and mutant CTPS. (A) Amino acid sequence alignment among hCTPS, mCTPS, and dCTPS. A partial view is presented here. (B) Immunostaining results of CTPS-TbID and CTPS H355A -TbID is shown in follicle cells. Scale bar, 10μm. (C) Ovaries were dissected from 14-day old flies and raised on either 100 μM biotin-containing food or regular food. Confocal images of labeled proteins detected by staining with streptavidin-Cy3 are presented, along with the expression of CTPS-TbID and CTPS H355A -TbID detected by anti-V5 blotting. All images were acquired from follicle cells. Scale bar, 10μm. (D) Streptavidin-HRP was used for the detection of labeled proteins, while anti-V5 antibody was used to detect the expression of CTPS-TbID and CTPS H355A –TbID. All ovaries samples were collected from 14-day old flies and CTPS-TbID and CTPS H355A –TbID were expressed using da-GAL4 driver in (B-D).
    Figure Legend Snippet: TurboID-mediated proximal biotinylation of CTPS cytoophidium and mutant CTPS. (A) Amino acid sequence alignment among hCTPS, mCTPS, and dCTPS. A partial view is presented here. (B) Immunostaining results of CTPS-TbID and CTPS H355A -TbID is shown in follicle cells. Scale bar, 10μm. (C) Ovaries were dissected from 14-day old flies and raised on either 100 μM biotin-containing food or regular food. Confocal images of labeled proteins detected by staining with streptavidin-Cy3 are presented, along with the expression of CTPS-TbID and CTPS H355A -TbID detected by anti-V5 blotting. All images were acquired from follicle cells. Scale bar, 10μm. (D) Streptavidin-HRP was used for the detection of labeled proteins, while anti-V5 antibody was used to detect the expression of CTPS-TbID and CTPS H355A –TbID. All ovaries samples were collected from 14-day old flies and CTPS-TbID and CTPS H355A –TbID were expressed using da-GAL4 driver in (B-D).

    Techniques Used: Mutagenesis, Sequencing, Immunostaining, Labeling, Staining, Expressing

    CTPS-mCherry was expressed ubiquitously via da-GAL4 driver. Western blotting visualizing biotinylated proteins in different tissues from adult flies with streptavidin-HRP. CTPS-mCherry expression was detected by anti-V5 blotting.
    Figure Legend Snippet: CTPS-mCherry was expressed ubiquitously via da-GAL4 driver. Western blotting visualizing biotinylated proteins in different tissues from adult flies with streptavidin-HRP. CTPS-mCherry expression was detected by anti-V5 blotting.

    Techniques Used: Western Blot, Expressing

    TurboID application in Drosophila . (A) CTPS-TbID and CTPS-miniTb containing V5 tag at C-terminal were cloned into pAc vectors and were transfected into Drosophila cultured S2 cells. Confocal images of cells are presented. DNA was labeled with Hoechst 33342 (blue). Scale bar, 10 μm. (B)Transgenic flies of UAS-CTPS-mCherry-V5 and UAS-CTPS-TbID-V5 were generated. Flies raised on either biotin-containing food or regular food from early embryo stages to larvae, pupae or adulthood, were collected and lysed and then blotted with Streptavidin-HRP to visualize biotinylated proteins. Anti-V5 antibody was used to detect the fused expression of CTPS-mCherry and CTPS-TbID, which was expressed ubiquitously via da-GAL4 driver. The molecular weight of CTPS-mCherry (98kD) is a little smaller than CTPS-TbID (105kD). (C) Both CTPS-mCherry and CTPS-TbID were specifically expressed in germline cells driven by nanos-GAL4. Ovaries from 14-day old flies grown on 100 μM biotin-containing food were dissected. Representative images of biotinylated proteins were obtained after detection by staining with streptavidin-488, while the expression of CTPS-mCherry and CTP-TbID was detected by anti-V5 blotting. Scale bar, 20 μm. (D) CTPS-TbID was expressed ubiquitously via da-GAL4 driver. Western blotting with streptavidin-HRP to visualize biotinylated proteins in different tissues from adult flies raised on either 100 μM biotin-containing food or regular food is presented. CTPS expression was detected by anti-V5 blotting.
    Figure Legend Snippet: TurboID application in Drosophila . (A) CTPS-TbID and CTPS-miniTb containing V5 tag at C-terminal were cloned into pAc vectors and were transfected into Drosophila cultured S2 cells. Confocal images of cells are presented. DNA was labeled with Hoechst 33342 (blue). Scale bar, 10 μm. (B)Transgenic flies of UAS-CTPS-mCherry-V5 and UAS-CTPS-TbID-V5 were generated. Flies raised on either biotin-containing food or regular food from early embryo stages to larvae, pupae or adulthood, were collected and lysed and then blotted with Streptavidin-HRP to visualize biotinylated proteins. Anti-V5 antibody was used to detect the fused expression of CTPS-mCherry and CTPS-TbID, which was expressed ubiquitously via da-GAL4 driver. The molecular weight of CTPS-mCherry (98kD) is a little smaller than CTPS-TbID (105kD). (C) Both CTPS-mCherry and CTPS-TbID were specifically expressed in germline cells driven by nanos-GAL4. Ovaries from 14-day old flies grown on 100 μM biotin-containing food were dissected. Representative images of biotinylated proteins were obtained after detection by staining with streptavidin-488, while the expression of CTPS-mCherry and CTP-TbID was detected by anti-V5 blotting. Scale bar, 20 μm. (D) CTPS-TbID was expressed ubiquitously via da-GAL4 driver. Western blotting with streptavidin-HRP to visualize biotinylated proteins in different tissues from adult flies raised on either 100 μM biotin-containing food or regular food is presented. CTPS expression was detected by anti-V5 blotting.

    Techniques Used: Clone Assay, Transfection, Cell Culture, Labeling, Generated, Expressing, Molecular Weight, Staining, Western Blot

    Model for TurboID-based labelling of CTPS cytoophidium proximate proteome. (A) TurboID was fused in-frame with wild type and mutant CTPS. Provided with biotin, TurboID can use biotin to biotinylate CTPS neighboring proteins. Cells are lysed and biotinylated proteins are captured using streptavidin beads. Subsequently, small peptides are generated by trypsin digestion and peptides are analyzed by mass spectrometry. Note that just a finite number of TurboID are shown in CTPS cytoophidium and disrupted cytoophidium. (B) Diagram of the expression cassettes used for the generation of transgenic flies. TurboID and V5 tag were fused to the C-terminal of wild type and mutant CTPS. mCherry was used as control. The same flexible linker was inserted into three cassettes.
    Figure Legend Snippet: Model for TurboID-based labelling of CTPS cytoophidium proximate proteome. (A) TurboID was fused in-frame with wild type and mutant CTPS. Provided with biotin, TurboID can use biotin to biotinylate CTPS neighboring proteins. Cells are lysed and biotinylated proteins are captured using streptavidin beads. Subsequently, small peptides are generated by trypsin digestion and peptides are analyzed by mass spectrometry. Note that just a finite number of TurboID are shown in CTPS cytoophidium and disrupted cytoophidium. (B) Diagram of the expression cassettes used for the generation of transgenic flies. TurboID and V5 tag were fused to the C-terminal of wild type and mutant CTPS. mCherry was used as control. The same flexible linker was inserted into three cassettes.

    Techniques Used: Mutagenesis, Generated, Mass Spectrometry, Expressing, Transgenic Assay

    23) Product Images from "Endothelial nitric oxide synthase protein distribution and nitric oxide production in endothelial cells along the coronary vascular tree"

    Article Title: Endothelial nitric oxide synthase protein distribution and nitric oxide production in endothelial cells along the coronary vascular tree

    Journal: Microvascular research

    doi: 10.1016/j.mvr.2018.11.004

    Isolation of coronary microvascular endothelial cells (MEC) with limited contamination from other cells types. Myocardial sections were digested and MEC isolated using biotinylated-anti-CD31 and streptavidin-coated paramagnetic beads. (A) Histological evaluation using Masson trichrome stain revealed that no vessels in the myocardial sections used exceeded ~300 μm luminal diameter and all but one vessel in each myocardial sample were under 150 μm diameter. Effectiveness of the methodology was determined by immunofluorescence and immunoblot to evaluate purity of the isolated MEC. (B-E) Hoechst-positive and CD31-positive images from MEC using epifluorescence microscopy were colocalized and a pixel fluorogram constructed. (F-G) Protein levels of eNOS, smooth muscle α-actin (SMA), and cardiac troponin I (cTnI) were compared by immunoblot analyses in lysates from endothelial cells (EC), small arteries (SA), arterioles (Art), and myocardium (M). EC1 EC2 are microvascular endothelial cell preps from two different pig hearts. These data demonstrate that 97% of cells isolated from the myocardial sample preparation were endothelial cells. Similar analyses in endothelial cells isolated from conduit arteries revealed comparable purity.
    Figure Legend Snippet: Isolation of coronary microvascular endothelial cells (MEC) with limited contamination from other cells types. Myocardial sections were digested and MEC isolated using biotinylated-anti-CD31 and streptavidin-coated paramagnetic beads. (A) Histological evaluation using Masson trichrome stain revealed that no vessels in the myocardial sections used exceeded ~300 μm luminal diameter and all but one vessel in each myocardial sample were under 150 μm diameter. Effectiveness of the methodology was determined by immunofluorescence and immunoblot to evaluate purity of the isolated MEC. (B-E) Hoechst-positive and CD31-positive images from MEC using epifluorescence microscopy were colocalized and a pixel fluorogram constructed. (F-G) Protein levels of eNOS, smooth muscle α-actin (SMA), and cardiac troponin I (cTnI) were compared by immunoblot analyses in lysates from endothelial cells (EC), small arteries (SA), arterioles (Art), and myocardium (M). EC1 EC2 are microvascular endothelial cell preps from two different pig hearts. These data demonstrate that 97% of cells isolated from the myocardial sample preparation were endothelial cells. Similar analyses in endothelial cells isolated from conduit arteries revealed comparable purity.

    Techniques Used: Isolation, Staining, Immunofluorescence, Epifluorescence Microscopy, Construct, Sample Prep

    24) Product Images from "3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids"

    Article Title: 3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

    Journal: Journal of structural biology

    doi: 10.1016/j.jsb.2019.06.004

    Processing of the streptavidin lattice in our images of Pol II EC(CPD) on streptavidin affinity support grids. (A) A representative micrograph showing Pol II EC(CPD) particles on a streptavidin crystal lattice. (B) FFT of the micrograph in (A) showing diffraction spots from the streptavidin crystal lattice. (C) ). Each diffraction spot is shown as a square and the size of the square indicates the signal to noise ratio for that spot. (D) The same micrograph shown in (A) after removing the streptavidin lattice using Fourier filtering. (E) FFT of the micrograph in (D) showing the disappearance of the diffraction spots. Zoom-ins of the regions highlighted in (A) and (D) (black squares), and (B) and (E) (white square) are shown below (for A and B) or above (for D and E) the corresponding panels. (F) Plot of the resolution at which the cross-correlation between the calculated and observed CTFs is 0.5 as a function of the estimated resolution of the streptavidin crystal. The grey horizontal line shows the CTF resolution cutoff used for image processing (4.5Å). All the micrographs above the grey line were discarded. The grey (no cutoff), orange (4.5Å cutoff) and red (2.9 Å cutoff) vertical dashed lines show the diffraction spot resolution cutoffs used for different reconstruction trials. Micrographs to the left of the dashed lines (shown by horizontal lines of the corresponding colors) were used for the reconstructions. The resolutions of the maps from these trials along with the number of particles contributing to the reconstructions are shown above the graph. The map reported in this work was the one obtained without imposing a diffraction resolution cutoff.
    Figure Legend Snippet: Processing of the streptavidin lattice in our images of Pol II EC(CPD) on streptavidin affinity support grids. (A) A representative micrograph showing Pol II EC(CPD) particles on a streptavidin crystal lattice. (B) FFT of the micrograph in (A) showing diffraction spots from the streptavidin crystal lattice. (C) ). Each diffraction spot is shown as a square and the size of the square indicates the signal to noise ratio for that spot. (D) The same micrograph shown in (A) after removing the streptavidin lattice using Fourier filtering. (E) FFT of the micrograph in (D) showing the disappearance of the diffraction spots. Zoom-ins of the regions highlighted in (A) and (D) (black squares), and (B) and (E) (white square) are shown below (for A and B) or above (for D and E) the corresponding panels. (F) Plot of the resolution at which the cross-correlation between the calculated and observed CTFs is 0.5 as a function of the estimated resolution of the streptavidin crystal. The grey horizontal line shows the CTF resolution cutoff used for image processing (4.5Å). All the micrographs above the grey line were discarded. The grey (no cutoff), orange (4.5Å cutoff) and red (2.9 Å cutoff) vertical dashed lines show the diffraction spot resolution cutoffs used for different reconstruction trials. Micrographs to the left of the dashed lines (shown by horizontal lines of the corresponding colors) were used for the reconstructions. The resolutions of the maps from these trials along with the number of particles contributing to the reconstructions are shown above the graph. The map reported in this work was the one obtained without imposing a diffraction resolution cutoff.

    Techniques Used:

    Biotinylation of an RNA Polymerase II Elongation Complex stalled at a cyclobutane pyrimidine dimer (CPD) lesion (Pol II EC(CPD)). (A) Schematic diagram of the assay used to determine the degree of biotinylation of Pol II EC(CPD). (B) ). (C) Negative stain EM micrographs of biotinylated Pol II EC(CPD) on continuous carbon (top) and streptavidin monolayer (bottom) grids.
    Figure Legend Snippet: Biotinylation of an RNA Polymerase II Elongation Complex stalled at a cyclobutane pyrimidine dimer (CPD) lesion (Pol II EC(CPD)). (A) Schematic diagram of the assay used to determine the degree of biotinylation of Pol II EC(CPD). (B) ). (C) Negative stain EM micrographs of biotinylated Pol II EC(CPD) on continuous carbon (top) and streptavidin monolayer (bottom) grids.

    Techniques Used: Staining

    3.1Å cryo-EM map of a randomly biotinylated Pol II EC(CPD) obtained using monolayer streptavidin crystal affinity grids. (A) Locally-filtered Cryo-EM map of Pol II EC(CPD). The map is segmented into the following regions: Pol II (grey), template strand (TS) (blue), non-template strand (NTS) (green) and RNA strand (red). The inset highlights two domains of Pol II we refer to in the text on a cartoon representation of the structure. (B–D) Representative regions of the Pol II EC(CPD) map: an alpha helix (B), a beta sheet (C), and Watson-Crick base pairing between the TS and the nascent RNA (D). The map is shown in transparent grey with the atomic model fitted into the density. The color code for the atomic model is the same as in A. (E) Pol II EC(CPD) map colored according to local resolution. The top panel shows the surface views of the map in different orientations and the bottom panel shows the corresponding cross-sections to highlight the resolutions in the interior of the map. (F) Schematic representations of the expected (top) and observed (bottom) positions of the bases in the transcription scaffold. The grey cylinder represents the bridge helix. For the observed (bottom) scaffold the dash indicates that the CPD lesion (orange) is connected to the TS by a phosphodiester bond. Bases that are disordered in our map are shown in lighter color. The 5’ and 3’ ends of the TS (blue) and NTS (green) are indicated. (G) Top view of the atomic model of the transcription scaffold. The bridge helix is shown for reference. The inset shows the CPD lesion fitted to the segmented TS density. αβ
    Figure Legend Snippet: 3.1Å cryo-EM map of a randomly biotinylated Pol II EC(CPD) obtained using monolayer streptavidin crystal affinity grids. (A) Locally-filtered Cryo-EM map of Pol II EC(CPD). The map is segmented into the following regions: Pol II (grey), template strand (TS) (blue), non-template strand (NTS) (green) and RNA strand (red). The inset highlights two domains of Pol II we refer to in the text on a cartoon representation of the structure. (B–D) Representative regions of the Pol II EC(CPD) map: an alpha helix (B), a beta sheet (C), and Watson-Crick base pairing between the TS and the nascent RNA (D). The map is shown in transparent grey with the atomic model fitted into the density. The color code for the atomic model is the same as in A. (E) Pol II EC(CPD) map colored according to local resolution. The top panel shows the surface views of the map in different orientations and the bottom panel shows the corresponding cross-sections to highlight the resolutions in the interior of the map. (F) Schematic representations of the expected (top) and observed (bottom) positions of the bases in the transcription scaffold. The grey cylinder represents the bridge helix. For the observed (bottom) scaffold the dash indicates that the CPD lesion (orange) is connected to the TS by a phosphodiester bond. Bases that are disordered in our map are shown in lighter color. The 5’ and 3’ ends of the TS (blue) and NTS (green) are indicated. (G) Top view of the atomic model of the transcription scaffold. The bridge helix is shown for reference. The inset shows the CPD lesion fitted to the segmented TS density. αβ

    Techniques Used:

    The accessibility of surface-exposed lysines correlates with particle orientation. (A) 3D FSC plot for the Pol II EC(CPD) reconstruction. (B) Euler angle distribution of the particles contributing to the reconstruction. (C–E) Solvent exposed surface area (SASA) of the lysine primary amines available for biotinylation. We used the atomic model of a previously published Pol II elongation complex (PDB ID: 1Y77) to represent the Pol II EC(CPD) in these panels. (C) A rare (top) and a common (bottom) view of Pol II EC(CPD) are shown along with the corresponding plots of Euler angle distribution. (D) This panel shows the surfaces of Pol II that must face the SA layer to lead to the views shown in (C). The primary amines of lysines are color-coded according to their solvent accessible surface area (SASA), with the least exposed amines shown in yellow and the most exposed ones in red. (E) This panel shows the same views shown in (D) but with a cutting plane adjusted such that only lysines whose biotin moieties would be capable of interacting with the streptavidin monolayer are shown. The cutting plane was set to ~29Å from the lysine’s amine, which is the length of a fully stretched biotinylation reagent molecule. The orientations and the cutting planes of the Pol II EC with respect the streptavidin monolayer (ochre rectangle) are shown next to each of the views in (D) and (E).
    Figure Legend Snippet: The accessibility of surface-exposed lysines correlates with particle orientation. (A) 3D FSC plot for the Pol II EC(CPD) reconstruction. (B) Euler angle distribution of the particles contributing to the reconstruction. (C–E) Solvent exposed surface area (SASA) of the lysine primary amines available for biotinylation. We used the atomic model of a previously published Pol II elongation complex (PDB ID: 1Y77) to represent the Pol II EC(CPD) in these panels. (C) A rare (top) and a common (bottom) view of Pol II EC(CPD) are shown along with the corresponding plots of Euler angle distribution. (D) This panel shows the surfaces of Pol II that must face the SA layer to lead to the views shown in (C). The primary amines of lysines are color-coded according to their solvent accessible surface area (SASA), with the least exposed amines shown in yellow and the most exposed ones in red. (E) This panel shows the same views shown in (D) but with a cutting plane adjusted such that only lysines whose biotin moieties would be capable of interacting with the streptavidin monolayer are shown. The cutting plane was set to ~29Å from the lysine’s amine, which is the length of a fully stretched biotinylation reagent molecule. The orientations and the cutting planes of the Pol II EC with respect the streptavidin monolayer (ochre rectangle) are shown next to each of the views in (D) and (E).

    Techniques Used:

    25) Product Images from "3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids"

    Article Title: 3.1Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

    Journal: Journal of structural biology

    doi: 10.1016/j.jsb.2019.06.004

    Processing of the streptavidin lattice in our images of Pol II EC(CPD) on streptavidin affinity support grids. (A) A representative micrograph showing Pol II EC(CPD) particles on a streptavidin crystal lattice. (B) FFT of the micrograph in (A) showing diffraction spots from the streptavidin crystal lattice. (C) ). Each diffraction spot is shown as a square and the size of the square indicates the signal to noise ratio for that spot. (D) The same micrograph shown in (A) after removing the streptavidin lattice using Fourier filtering. (E) FFT of the micrograph in (D) showing the disappearance of the diffraction spots. Zoom-ins of the regions highlighted in (A) and (D) (black squares), and (B) and (E) (white square) are shown below (for A and B) or above (for D and E) the corresponding panels. (F) Plot of the resolution at which the cross-correlation between the calculated and observed CTFs is 0.5 as a function of the estimated resolution of the streptavidin crystal. The grey horizontal line shows the CTF resolution cutoff used for image processing (4.5Å). All the micrographs above the grey line were discarded. The grey (no cutoff), orange (4.5Å cutoff) and red (2.9 Å cutoff) vertical dashed lines show the diffraction spot resolution cutoffs used for different reconstruction trials. Micrographs to the left of the dashed lines (shown by horizontal lines of the corresponding colors) were used for the reconstructions. The resolutions of the maps from these trials along with the number of particles contributing to the reconstructions are shown above the graph. The map reported in this work was the one obtained without imposing a diffraction resolution cutoff.
    Figure Legend Snippet: Processing of the streptavidin lattice in our images of Pol II EC(CPD) on streptavidin affinity support grids. (A) A representative micrograph showing Pol II EC(CPD) particles on a streptavidin crystal lattice. (B) FFT of the micrograph in (A) showing diffraction spots from the streptavidin crystal lattice. (C) ). Each diffraction spot is shown as a square and the size of the square indicates the signal to noise ratio for that spot. (D) The same micrograph shown in (A) after removing the streptavidin lattice using Fourier filtering. (E) FFT of the micrograph in (D) showing the disappearance of the diffraction spots. Zoom-ins of the regions highlighted in (A) and (D) (black squares), and (B) and (E) (white square) are shown below (for A and B) or above (for D and E) the corresponding panels. (F) Plot of the resolution at which the cross-correlation between the calculated and observed CTFs is 0.5 as a function of the estimated resolution of the streptavidin crystal. The grey horizontal line shows the CTF resolution cutoff used for image processing (4.5Å). All the micrographs above the grey line were discarded. The grey (no cutoff), orange (4.5Å cutoff) and red (2.9 Å cutoff) vertical dashed lines show the diffraction spot resolution cutoffs used for different reconstruction trials. Micrographs to the left of the dashed lines (shown by horizontal lines of the corresponding colors) were used for the reconstructions. The resolutions of the maps from these trials along with the number of particles contributing to the reconstructions are shown above the graph. The map reported in this work was the one obtained without imposing a diffraction resolution cutoff.

    Techniques Used:

    Biotinylation of an RNA Polymerase II Elongation Complex stalled at a cyclobutane pyrimidine dimer (CPD) lesion (Pol II EC(CPD)). (A) Schematic diagram of the assay used to determine the degree of biotinylation of Pol II EC(CPD). (B) ). (C) Negative stain EM micrographs of biotinylated Pol II EC(CPD) on continuous carbon (top) and streptavidin monolayer (bottom) grids.
    Figure Legend Snippet: Biotinylation of an RNA Polymerase II Elongation Complex stalled at a cyclobutane pyrimidine dimer (CPD) lesion (Pol II EC(CPD)). (A) Schematic diagram of the assay used to determine the degree of biotinylation of Pol II EC(CPD). (B) ). (C) Negative stain EM micrographs of biotinylated Pol II EC(CPD) on continuous carbon (top) and streptavidin monolayer (bottom) grids.

    Techniques Used: Staining

    3.1Å cryo-EM map of a randomly biotinylated Pol II EC(CPD) obtained using monolayer streptavidin crystal affinity grids. (A) Locally-filtered Cryo-EM map of Pol II EC(CPD). The map is segmented into the following regions: Pol II (grey), template strand (TS) (blue), non-template strand (NTS) (green) and RNA strand (red). The inset highlights two domains of Pol II we refer to in the text on a cartoon representation of the structure. (B–D) Representative regions of the Pol II EC(CPD) map: an alpha helix (B), a beta sheet (C), and Watson-Crick base pairing between the TS and the nascent RNA (D). The map is shown in transparent grey with the atomic model fitted into the density. The color code for the atomic model is the same as in A. (E) Pol II EC(CPD) map colored according to local resolution. The top panel shows the surface views of the map in different orientations and the bottom panel shows the corresponding cross-sections to highlight the resolutions in the interior of the map. (F) Schematic representations of the expected (top) and observed (bottom) positions of the bases in the transcription scaffold. The grey cylinder represents the bridge helix. For the observed (bottom) scaffold the dash indicates that the CPD lesion (orange) is connected to the TS by a phosphodiester bond. Bases that are disordered in our map are shown in lighter color. The 5’ and 3’ ends of the TS (blue) and NTS (green) are indicated. (G) Top view of the atomic model of the transcription scaffold. The bridge helix is shown for reference. The inset shows the CPD lesion fitted to the segmented TS density. αβ
    Figure Legend Snippet: 3.1Å cryo-EM map of a randomly biotinylated Pol II EC(CPD) obtained using monolayer streptavidin crystal affinity grids. (A) Locally-filtered Cryo-EM map of Pol II EC(CPD). The map is segmented into the following regions: Pol II (grey), template strand (TS) (blue), non-template strand (NTS) (green) and RNA strand (red). The inset highlights two domains of Pol II we refer to in the text on a cartoon representation of the structure. (B–D) Representative regions of the Pol II EC(CPD) map: an alpha helix (B), a beta sheet (C), and Watson-Crick base pairing between the TS and the nascent RNA (D). The map is shown in transparent grey with the atomic model fitted into the density. The color code for the atomic model is the same as in A. (E) Pol II EC(CPD) map colored according to local resolution. The top panel shows the surface views of the map in different orientations and the bottom panel shows the corresponding cross-sections to highlight the resolutions in the interior of the map. (F) Schematic representations of the expected (top) and observed (bottom) positions of the bases in the transcription scaffold. The grey cylinder represents the bridge helix. For the observed (bottom) scaffold the dash indicates that the CPD lesion (orange) is connected to the TS by a phosphodiester bond. Bases that are disordered in our map are shown in lighter color. The 5’ and 3’ ends of the TS (blue) and NTS (green) are indicated. (G) Top view of the atomic model of the transcription scaffold. The bridge helix is shown for reference. The inset shows the CPD lesion fitted to the segmented TS density. αβ

    Techniques Used:

    The accessibility of surface-exposed lysines correlates with particle orientation. (A) 3D FSC plot for the Pol II EC(CPD) reconstruction. (B) Euler angle distribution of the particles contributing to the reconstruction. (C–E) Solvent exposed surface area (SASA) of the lysine primary amines available for biotinylation. We used the atomic model of a previously published Pol II elongation complex (PDB ID: 1Y77) to represent the Pol II EC(CPD) in these panels. (C) A rare (top) and a common (bottom) view of Pol II EC(CPD) are shown along with the corresponding plots of Euler angle distribution. (D) This panel shows the surfaces of Pol II that must face the SA layer to lead to the views shown in (C). The primary amines of lysines are color-coded according to their solvent accessible surface area (SASA), with the least exposed amines shown in yellow and the most exposed ones in red. (E) This panel shows the same views shown in (D) but with a cutting plane adjusted such that only lysines whose biotin moieties would be capable of interacting with the streptavidin monolayer are shown. The cutting plane was set to ~29Å from the lysine’s amine, which is the length of a fully stretched biotinylation reagent molecule. The orientations and the cutting planes of the Pol II EC with respect the streptavidin monolayer (ochre rectangle) are shown next to each of the views in (D) and (E).
    Figure Legend Snippet: The accessibility of surface-exposed lysines correlates with particle orientation. (A) 3D FSC plot for the Pol II EC(CPD) reconstruction. (B) Euler angle distribution of the particles contributing to the reconstruction. (C–E) Solvent exposed surface area (SASA) of the lysine primary amines available for biotinylation. We used the atomic model of a previously published Pol II elongation complex (PDB ID: 1Y77) to represent the Pol II EC(CPD) in these panels. (C) A rare (top) and a common (bottom) view of Pol II EC(CPD) are shown along with the corresponding plots of Euler angle distribution. (D) This panel shows the surfaces of Pol II that must face the SA layer to lead to the views shown in (C). The primary amines of lysines are color-coded according to their solvent accessible surface area (SASA), with the least exposed amines shown in yellow and the most exposed ones in red. (E) This panel shows the same views shown in (D) but with a cutting plane adjusted such that only lysines whose biotin moieties would be capable of interacting with the streptavidin monolayer are shown. The cutting plane was set to ~29Å from the lysine’s amine, which is the length of a fully stretched biotinylation reagent molecule. The orientations and the cutting planes of the Pol II EC with respect the streptavidin monolayer (ochre rectangle) are shown next to each of the views in (D) and (E).

    Techniques Used:

    26) Product Images from "A HSP60-targeting peptide for cell apoptosis imaging"

    Article Title: A HSP60-targeting peptide for cell apoptosis imaging

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2016.14

    Identification of HSP60 as the target of P17. ( a ) Protocols used for pull-down-assay-based analysis of binder of P17. ( b ) Biotin-P17 labeled streptavidin magnetic beads were used for pull-down assay from TRAIL-treated Jurkat cell extracts. Bound proteins were separated by SDS–PAGE. Absent from TRAIL-untreated and bead-control samples, a major band at 58 kDa was excised for mass spectrometry and identified as HSP60. ( c ) Mass spectrometry analysis identified peptides in HSP60 with 37% sequence coverage. Matched amino acids were shown in red. ( d ) Colocalization of FITC-P17 and anti-HSP60 (Alexa Fluor 594-conjugated) in TRAIL-treated Jurkat cells (Scale bars: 10 μm) observed by confocal microscopy (upper) and analyzed by Image-Pro Plus (bottom). ( e ) The schematic illustration for pull-down and western blot analysis. ( f ) Detection of HSP60 in pull-down samples by western blot and coomassie brilliant blue staining.
    Figure Legend Snippet: Identification of HSP60 as the target of P17. ( a ) Protocols used for pull-down-assay-based analysis of binder of P17. ( b ) Biotin-P17 labeled streptavidin magnetic beads were used for pull-down assay from TRAIL-treated Jurkat cell extracts. Bound proteins were separated by SDS–PAGE. Absent from TRAIL-untreated and bead-control samples, a major band at 58 kDa was excised for mass spectrometry and identified as HSP60. ( c ) Mass spectrometry analysis identified peptides in HSP60 with 37% sequence coverage. Matched amino acids were shown in red. ( d ) Colocalization of FITC-P17 and anti-HSP60 (Alexa Fluor 594-conjugated) in TRAIL-treated Jurkat cells (Scale bars: 10 μm) observed by confocal microscopy (upper) and analyzed by Image-Pro Plus (bottom). ( e ) The schematic illustration for pull-down and western blot analysis. ( f ) Detection of HSP60 in pull-down samples by western blot and coomassie brilliant blue staining.

    Techniques Used: Pull Down Assay, Labeling, Magnetic Beads, SDS Page, Mass Spectrometry, Sequencing, Confocal Microscopy, Western Blot, Staining

    27) Product Images from "Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue"

    Article Title: Hybrid Capture and Next-Generation Sequencing Identify Viral Integration Sites from Formalin-Fixed, Paraffin-Embedded Tissue

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.01.006

    Validating biotin-14-dCTP incorporation by a bind and boil method. The biotin–streptavidin dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.
    Figure Legend Snippet: Validating biotin-14-dCTP incorporation by a bind and boil method. The biotin–streptavidin dissociation constant (kD) is on the order of 4 × 10 −14 mol/L. However, boiling the biotin-steptavidin complexes in the presence of SDS releases these noncovalent complexes. To validate the incorporation of biotin-14-dCTP during the PCR, we assayed the supernatant of our PCR “bait” solution after mixing with Dynal M-280 beads. If biotin-14-dCTP did not incorporate during the PCR, we would expect the supernatant to contain PCR-amplified DNA. To assay the supernatant, we performed a buffer exchange using AmpureXP beads, eluted in water, and evaluated the supernatant of PCR on the Agilent BioAnalyzer 2100 High Sensitivity DNA chip (red dotted line). The Dynal M-280 beads were boiled in 0.1% SDS, and this supernatant was assayed for PCR products (dotted blue line). A second boil treatment was performed, and this supernatant was assayed for the presence of PCR products (dotted green line). FU, fluorescent units; nt, nucleotides.

    Techniques Used: Polymerase Chain Reaction, Amplification, Buffer Exchange, Chromatin Immunoprecipitation

    Pictorial representation of Washington University Capture. Washington University Capture (WUCap) enables solution-phase hybridization between double-stranded DNA PCR “bait” and whole-genome shotgun libraries. The solution-phase method we have developed for hybrid capture is robust and involves only a few basic steps. The bait used for targeting is dictated by primer-specific amplification of genomic targets generated during the PCR. Subsequently, the amplicons are used as a template in a second PCR incorporating biotin-14-dCTP. Genomic DNA is prepared from each of the samples to be sequenced, sheared to an average fragment size of 300 bp, enzymatically repaired to blunt the ends, and ligated to Illumina adapter sequences (at both ends). Five hundred nanograms of genomic DNA library is denatured, combined with 100 ng of the biotinylated “bait,” and hybridized for 48 hours. Mixing this hybridization reaction with streptavidin-coated superparamagnetic beads allows binding of biotinylated bait–target hybrids and selective removal from solution by applying a magnet field. The remaining supernatant is removed, and the beads are washed, removing nonspecific DNA. The enriched target sequences are released from the bead-bound bait sequences by denaturation (0.125 N NaOH), neutralized, amplified in the PCR to generate double-stranded Illumina libraries, and then sequenced.
    Figure Legend Snippet: Pictorial representation of Washington University Capture. Washington University Capture (WUCap) enables solution-phase hybridization between double-stranded DNA PCR “bait” and whole-genome shotgun libraries. The solution-phase method we have developed for hybrid capture is robust and involves only a few basic steps. The bait used for targeting is dictated by primer-specific amplification of genomic targets generated during the PCR. Subsequently, the amplicons are used as a template in a second PCR incorporating biotin-14-dCTP. Genomic DNA is prepared from each of the samples to be sequenced, sheared to an average fragment size of 300 bp, enzymatically repaired to blunt the ends, and ligated to Illumina adapter sequences (at both ends). Five hundred nanograms of genomic DNA library is denatured, combined with 100 ng of the biotinylated “bait,” and hybridized for 48 hours. Mixing this hybridization reaction with streptavidin-coated superparamagnetic beads allows binding of biotinylated bait–target hybrids and selective removal from solution by applying a magnet field. The remaining supernatant is removed, and the beads are washed, removing nonspecific DNA. The enriched target sequences are released from the bead-bound bait sequences by denaturation (0.125 N NaOH), neutralized, amplified in the PCR to generate double-stranded Illumina libraries, and then sequenced.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Amplification, Generated, Binding Assay

    28) Product Images from "Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ"

    Article Title: Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ

    Journal: Oncogene

    doi: 10.1038/onc.2013.594

    Wig-1 binds to the 3′-UTR of FAS mRNA and regulates its stability through the ARE. RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b (Flag) or pCMVtag2bhWig-1 (Flag-Wig-1) and in Saos-2 TetON cells without insert (Ctrl) or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 (Wig-1) or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA (Wig-1ZF1pm) ( a ). Wig-1 was precipitated with anti-Flag beads, and bound RNA was purified and quantified by qRT–PCR. GAPDH mRNA levels were used as internal control. Labeling of nascent RNA with 4-sU, RNA extraction, conjugation of the 4-sU to biotin and separation of nascent and older RNA with streptavidin beads followed by qRT–PCR analysis of the two populations separately showed no significant variation in the 4-sU-labeled RNA, whereas we observed an increase in the older, unlabeled RNA population after Wig-1 knockdown. GAPDH was used as internal control ( b ). Biotin pull-down assay using FAS 5′-UTR-ORF, FAS 3′-UTR, FAS 3′-UTR distal and FAS 3′-UTR distal dARE probes ( c ) followed by western blotting for Wig-1 shows that Wig-1 binds to the ARE in the 3′-UTR of FAS. ( d ). Representative image from one of two independent experiments. To determine whether the ARE is required for Wig-1 regulation of FAS mRNA, we generated the constructs 2–5 described in ( c ) or the p53 3′-UTR lacking the ARE as negative control and cloned them downstream of the Renilla reporter into the psiCheck-2 vector. Luciferase assays confirmed that the ARE in the 3′-UTR of FAS mRNA is essential for regulation by Wig-1, as constructs lacking this ARE do not show increased activity after Wig-1 depletion ( e ). Columns and error bars in ( a ), ( b ) and ( e ) represent the mean±s.d.; n =3; *** P
    Figure Legend Snippet: Wig-1 binds to the 3′-UTR of FAS mRNA and regulates its stability through the ARE. RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b (Flag) or pCMVtag2bhWig-1 (Flag-Wig-1) and in Saos-2 TetON cells without insert (Ctrl) or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 (Wig-1) or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA (Wig-1ZF1pm) ( a ). Wig-1 was precipitated with anti-Flag beads, and bound RNA was purified and quantified by qRT–PCR. GAPDH mRNA levels were used as internal control. Labeling of nascent RNA with 4-sU, RNA extraction, conjugation of the 4-sU to biotin and separation of nascent and older RNA with streptavidin beads followed by qRT–PCR analysis of the two populations separately showed no significant variation in the 4-sU-labeled RNA, whereas we observed an increase in the older, unlabeled RNA population after Wig-1 knockdown. GAPDH was used as internal control ( b ). Biotin pull-down assay using FAS 5′-UTR-ORF, FAS 3′-UTR, FAS 3′-UTR distal and FAS 3′-UTR distal dARE probes ( c ) followed by western blotting for Wig-1 shows that Wig-1 binds to the ARE in the 3′-UTR of FAS. ( d ). Representative image from one of two independent experiments. To determine whether the ARE is required for Wig-1 regulation of FAS mRNA, we generated the constructs 2–5 described in ( c ) or the p53 3′-UTR lacking the ARE as negative control and cloned them downstream of the Renilla reporter into the psiCheck-2 vector. Luciferase assays confirmed that the ARE in the 3′-UTR of FAS mRNA is essential for regulation by Wig-1, as constructs lacking this ARE do not show increased activity after Wig-1 depletion ( e ). Columns and error bars in ( a ), ( b ) and ( e ) represent the mean±s.d.; n =3; *** P

    Techniques Used: Immunoprecipitation, Transfection, Stable Transfection, Mutagenesis, Purification, Quantitative RT-PCR, Labeling, RNA Extraction, Conjugation Assay, Pull Down Assay, Western Blot, Generated, Construct, Negative Control, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay

    29) Product Images from "Actin-Dependent Nonlytic Rotavirus Exit and Infectious Virus Morphogenetic Pathway in Nonpolarized Cells"

    Article Title: Actin-Dependent Nonlytic Rotavirus Exit and Infectious Virus Morphogenetic Pathway in Nonpolarized Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.02076-17

    VP4 present at the plasma membrane is incorporated into viral particles. MA104 cells were infected with RRV (at an MOI of 3 FFU/cell), and at 6 hpi the cells were biotinylated with the impermeant reagent sulfo-NHS-LC-biotin for 30 min. The cells were then washed and incubated until 12 hpi. At this time, TLPs were purified twice by use of CsCl isopycnic gradients. (A) TLPs obtained from biotinylated (Biot) and nonbiotinylated (Ctrl) cells were purified by use of streptavidin (Strp) magnetic beads and analyzed by Western blotting using the anti-TLP antibody. (B) Control TLPs (1) and biotinylated TLPs (2) were stained with streptavidin-gold and analyzed by electron microscopy as described in Materials and Methods.
    Figure Legend Snippet: VP4 present at the plasma membrane is incorporated into viral particles. MA104 cells were infected with RRV (at an MOI of 3 FFU/cell), and at 6 hpi the cells were biotinylated with the impermeant reagent sulfo-NHS-LC-biotin for 30 min. The cells were then washed and incubated until 12 hpi. At this time, TLPs were purified twice by use of CsCl isopycnic gradients. (A) TLPs obtained from biotinylated (Biot) and nonbiotinylated (Ctrl) cells were purified by use of streptavidin (Strp) magnetic beads and analyzed by Western blotting using the anti-TLP antibody. (B) Control TLPs (1) and biotinylated TLPs (2) were stained with streptavidin-gold and analyzed by electron microscopy as described in Materials and Methods.

    Techniques Used: Infection, Incubation, Purification, Magnetic Beads, Western Blot, Staining, Electron Microscopy

    Transport of VP4 to the plasma membrane is affected by JAS treatment. MA104 cells were infected with RRV (at an MOI of 3) and left untreated or treated with JAS (1 μM) for 6 h before being biotinylated. At the indicated times postinfection, the cells were biotinylated as described in Materials and Methods. The biotinylated proteins were precipitated by use of streptavidin (Strp) magnetic beads and analyzed by Western blotting using the anti-TLP antibody. The loading control was developed with a streptavidin-peroxidase conjugate. (A) Representative Western blot of proteins harvested at 6 hpi. A biotinylated membrane protein was used as a loading control. (B) Quantification of the biotinylated VP4 present at the plasma membrane by densitometry analysis as described in Materials and Methods. The amount of VP4 on the surfaces of control untreated cells at each time postinfection was taken as 100%. The arithmetic means and standard deviations for three independent experiments are shown. **, P
    Figure Legend Snippet: Transport of VP4 to the plasma membrane is affected by JAS treatment. MA104 cells were infected with RRV (at an MOI of 3) and left untreated or treated with JAS (1 μM) for 6 h before being biotinylated. At the indicated times postinfection, the cells were biotinylated as described in Materials and Methods. The biotinylated proteins were precipitated by use of streptavidin (Strp) magnetic beads and analyzed by Western blotting using the anti-TLP antibody. The loading control was developed with a streptavidin-peroxidase conjugate. (A) Representative Western blot of proteins harvested at 6 hpi. A biotinylated membrane protein was used as a loading control. (B) Quantification of the biotinylated VP4 present at the plasma membrane by densitometry analysis as described in Materials and Methods. The amount of VP4 on the surfaces of control untreated cells at each time postinfection was taken as 100%. The arithmetic means and standard deviations for three independent experiments are shown. **, P

    Techniques Used: Infection, Magnetic Beads, Western Blot

    Related Articles

    Incubation:

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection
    Article Snippet: .. For bead capture, 500 ng of the resulting mixture was incubated with 25 μ L of streptavidin beads resuspended in 2× binding/washing buffer, with the rest reserved for solid-state (SS)-nanopore measurements. ..

    Article Title: miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family
    Article Snippet: .. 90% of cell lysate was incubated with streptavidin magnetic beads (New England Biolabs) for 6 hours at 4°C and 10% of cell lysate was used for input RNA. ..

    Binding Assay:

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection
    Article Snippet: .. For bead capture, 500 ng of the resulting mixture was incubated with 25 μ L of streptavidin beads resuspended in 2× binding/washing buffer, with the rest reserved for solid-state (SS)-nanopore measurements. ..

    Magnetic Beads:

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding
    Article Snippet: .. Streptavidin magnetic beads (10 µL, New England BioLabs) were used to precipitate biotinylated species on a magnetic tube rack. .. Specific protein targets were detected using primary antibodies followed by membrane stripping before detection of overall biotinylated proteins.

    Article Title: Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB
    Article Snippet: .. 80 μl of pre-washed streptavidin magnetic beads (NEB) were added, mixed and rotated at room temperature for 10 minutes. ..

    Article Title: The Conserved C-Terminus of the PcrA/UvrD Helicase Interacts Directly with RNA Polymerase
    Article Snippet: .. Pull down experiments Pull down experiments were carried out using streptavidin magnetic beads (New England Biolabs). .. Each addition or washing step was performed at 4 °C by placing Eppendorf tubes containing beads on a rotator device, designed to continually but gently mix the beads with the sample.

    Article Title: miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family
    Article Snippet: .. 90% of cell lysate was incubated with streptavidin magnetic beads (New England Biolabs) for 6 hours at 4°C and 10% of cell lysate was used for input RNA. ..

    Article Title: Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions
    Article Snippet: .. Bead washing Aliquots of 20 μl of Streptavidin magnetic beads (New England Biolabs, S1420S) were pre-washed three times with 2× BW buffer ( ); resuspended in 50 μl 2× BW; mixed with the 50 μl SPEX primer extension reaction and rotated at room temperature for 30 min to immobilize biotinylated molecules to the beads; then a series of washes with 2× BW, 0.15 M NaOH and 1× Tris/EDTA (TE, pH 7.5) were carried out as described by Chen et al. ( ) to remove everything but biotinylated molecules. .. The beads were resuspended to 14 μl with 0.1× Qiagen buffer EB (10 mM Tris·Cl, pH 8.5).

    Article Title: Aberrant Glycosylation in the Human Trabecular Meshwork
    Article Snippet: .. The precipitate was recovered with 25 μl of 4μg/μl streptavidin coupled magnetic beads (cat# S1420S, New England Biolabs, Ipswitch, MA). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs streptavidin magnetic beads
    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through <t>streptavidin</t> purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.
    Streptavidin Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin magnetic beads/product/New England Biolabs
    Average 99 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    streptavidin magnetic beads - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    98
    New England Biolabs hydrophilic streptavidin magnetic beads
    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the <t>streptavidin</t> beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value
    Hydrophilic Streptavidin Magnetic Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hydrophilic streptavidin magnetic beads/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hydrophilic streptavidin magnetic beads - by Bioz Stars, 2020-09
    98/100 stars
      Buy from Supplier

    92
    beckman coulter streptavidin magnetic beads
    Physical file separations in DENSE storage rescue the decreased sequencing efficiency experienced by high-capacity databases. a , A library of five files was ordered and analyzed using NGS to confirm an even file distribution. b , File 3 strands were enriched over increasingly higher capacity backgrounds of non-specific DNA strands using 30 cycles of random-access PCR. Random access failed to enrich File 3 to above 50% of the total sample once the background capacity reached 31.1 GB, as measured by quantitative PCR. c , DENSE physically extracts a file (orange) from the database so only its strands are sequenced. A primer functionalized with a chemical handle (yellow diamond) is used to execute one emulsion PCR cycle to create chemically labeled copies of the desired file’s strands. Functionalized magnetic beads (brown) that bind to the chemical handle are added to the sample. The desired file is bound to the bead, and the unbound solution containing the original database is removed and saved for future reuse. The bound file is then eluted from the bead. d , After <t>biotin-streptavidin</t> file extractions, the remaining solution still contained all files while the target files were enriched and physically separated, as measured by next generation sequencing. By mapping sequencing reads to the original file sequences, all targeted data were confirmed recovered. The target file was retained in the supernatant containing the database and was able to be copied and extracted again. File 1 was extracted three sequential times, and File 2 was extracted from the solution remaining after an initial extraction of File 1. e , File extractions using fluorescein, digoxigenin, and polyA(25) as chemical handles also successfully separated target files from the database. f , A large-scale background mimicking diverse data was created using error prone PCR 13 to mutagenize and amplify File 1. g , Random access was compared directly to chemical handle extractions. File 3 strands, with a starting fraction of 0.03% of the total number of strands, were enriched over a high-capacity background equivalent to 5.53 TB of undesired, non-specific strands using either random access (black) or PCR followed by chemical handle primer extractions (blue, green, purple or pink). After 5, 15, and 30 cycles of PCR (random access), enrichment of File 3 was 0.0%, 0.0%, and 1.69% of the total sample, respectively. After biotin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 87.5%, and 100% of the total sample, respectively. After fluorescein-modified PCR followed by extraction, the enrichment of File 3 was 0.1%, 49.6%, and 100% of the total sample, respectively. After digoxigenin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 14.2%, and 100% of the total sample, respectively. After poly(A)-25-modified PCR followed by extraction, the enrichment of File 3 was 0.09%, 0.47%, and 100% of the total sample, respectively.
    Streptavidin Magnetic Beads, supplied by beckman coulter, used in various techniques. Bioz Stars score: 92/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin magnetic beads/product/beckman coulter
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin magnetic beads - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Journal: Scientific Reports

    Article Title: Src kinase phosphorylates Notch1 to inhibit MAML binding

    doi: 10.1038/s41598-018-33920-y

    Figure Lengend Snippet: Phosphorylation of tyrosine residues on the N1ICD are SFK dependent. ( A ) Diagram of BioID experiment. In the presence of biotin, the NICD::BirA fusion protein biotinylates nearby proteins which can be affinity captured through streptavidin purification. ( B ) Western blot analysis of affinity captured material from BioID experiment using a N1ICD::BirA-HA fusion protein in 293 T cells. Expression of Src variants is denoted as WT = wild-type, CA = constitutively active, and DN = dominant negative. ( C ) Amino acid sequence of 2058–2161::BirA-HA fusion protein containing a 104 amino acid fragment of N1ICD fused to a biotin ligase. ( D ) Western blot analysis of affinity captured material from BioID experiment using the 2058–2161::BirA-HA fusion in 293 T cells. ( E ) Western blot analysis of immunoprecipitated N1ICD in the presence or absence of c-Src overexpression in 293 T cells. ( F ) Western blot analysis of immunoprecipitated N1ICD under conditions SFK inhibition (AZM475271) and control in 293 T cells. ( G ) Western blot analysis of immunoprecipitated N1ICD and N4ICD in the presence or absence of c-Src overexpression in 293 T cells. In all panels, western blots depict representative images from experiments that were replicated at least three independent times.

    Article Snippet: Streptavidin magnetic beads (10 µL, New England BioLabs) were used to precipitate biotinylated species on a magnetic tube rack.

    Techniques: Purification, Western Blot, Expressing, Dominant Negative Mutation, Sequencing, Immunoprecipitation, Over Expression, Inhibition

    (a) Cut-away diagram of the crystal structure of WT streptavidin (Protein Database structure 1MK5) showing that its biotin-binding regions are highly hydrophobic in their active state. Scale goes from hydrophilic (red) to hydrophobic (green), (b) Liquid-phase elution. Biotinylated DNA–MS complexes (in water) are added to a combination of phenol/chloroform/isoamyl alcohol (yellow) with water (blue) and mixed. As proteins are exposed to phenol (center), they dissociate from the DNA and then segregate into the organic when the phases are allowed to separate (right), (c) Gel analysis of liquid-phase elution. In the first panel, lane 1 is the 150 bp DNA construct alone, lane 2 is the construct bound to MS, lane 3 is the recovered DNA following elution, and lane 4 is the recovered DNA bound to fresh MS. In the second panel, the left lane shows elution with pure chloroform (no phenol), and the right lane shows elution with pure water at room temperature.

    Journal: Analytical chemistry

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection

    doi: 10.1021/acs.analchem.9b01873

    Figure Lengend Snippet: (a) Cut-away diagram of the crystal structure of WT streptavidin (Protein Database structure 1MK5) showing that its biotin-binding regions are highly hydrophobic in their active state. Scale goes from hydrophilic (red) to hydrophobic (green), (b) Liquid-phase elution. Biotinylated DNA–MS complexes (in water) are added to a combination of phenol/chloroform/isoamyl alcohol (yellow) with water (blue) and mixed. As proteins are exposed to phenol (center), they dissociate from the DNA and then segregate into the organic when the phases are allowed to separate (right), (c) Gel analysis of liquid-phase elution. In the first panel, lane 1 is the 150 bp DNA construct alone, lane 2 is the construct bound to MS, lane 3 is the recovered DNA following elution, and lane 4 is the recovered DNA bound to fresh MS. In the second panel, the left lane shows elution with pure chloroform (no phenol), and the right lane shows elution with pure water at room temperature.

    Article Snippet: For bead capture, 500 ng of the resulting mixture was incubated with 25 μ L of streptavidin beads resuspended in 2× binding/washing buffer, with the rest reserved for solid-state (SS)-nanopore measurements.

    Techniques: Binding Assay, Construct

    Gel analysis of 150 bp biotinylated DNA elution from streptavidin-conjugated magnetic beads. Lanes from left to right show: unprocessed DNA; fresh DNA bound to and eluted from fresh beads; recovered DNA bound to and eluted from fresh beads; recovered DNA bound to and eluted from used beads; and fresh DNA bound to and eluted from used beads. All elutions performed with 12.5% (v/v) phenol at room temperature.

    Journal: Analytical chemistry

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection

    doi: 10.1021/acs.analchem.9b01873

    Figure Lengend Snippet: Gel analysis of 150 bp biotinylated DNA elution from streptavidin-conjugated magnetic beads. Lanes from left to right show: unprocessed DNA; fresh DNA bound to and eluted from fresh beads; recovered DNA bound to and eluted from fresh beads; recovered DNA bound to and eluted from used beads; and fresh DNA bound to and eluted from used beads. All elutions performed with 12.5% (v/v) phenol at room temperature.

    Article Snippet: For bead capture, 500 ng of the resulting mixture was incubated with 25 μ L of streptavidin beads resuspended in 2× binding/washing buffer, with the rest reserved for solid-state (SS)-nanopore measurements.

    Techniques: Magnetic Beads

    (a) Schematic showing isolation of biotinylated DNA. A mixture of biotinylated (red) and nonbiotinylated (blue) DNA fragments are incubated with streptavidin magnetic beads (l). The beads with bound DNA are collected magnetically, and nonbiotinylated DNA is washed away (2). Biotinylated DNA is eluted with 12.5% (v/v) phenol (3). (b) Gel analysis showing: unprocessed 48.5 kbp (i) λ -phage DNA (lane l); λ -phage DNA biotinylated at one end digested with PspXI to produce fragments approximately 33.5 (ii) and 15 kbp (iii) in length (lane 2); and the fragments after isolation and phenol elution from streptavidin beads (lane 3). The red arrow indicates the biotinylated ~15 kbp fragment, (c) Normalized SS-nanopore event histograms of ECD for the initial admixture (top, n = 1158) and the product of bead isolation (bottom, n = 519). SS-nanopore diameters are 6.1 and 6.1 nm, respectively. Lower ECD corresponds to lower molecular weight (i.e., the 15 kbp biotinylated DNA). Insets show a typical conductance trace for each measurement, with initial (open pore) conductance to the left and after addition of the DNA to the right. Spikes indicate molecular translocations. Scale bars are 500 ms (horizontal) and 1 nS (vertical).

    Journal: Analytical chemistry

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection

    doi: 10.1021/acs.analchem.9b01873

    Figure Lengend Snippet: (a) Schematic showing isolation of biotinylated DNA. A mixture of biotinylated (red) and nonbiotinylated (blue) DNA fragments are incubated with streptavidin magnetic beads (l). The beads with bound DNA are collected magnetically, and nonbiotinylated DNA is washed away (2). Biotinylated DNA is eluted with 12.5% (v/v) phenol (3). (b) Gel analysis showing: unprocessed 48.5 kbp (i) λ -phage DNA (lane l); λ -phage DNA biotinylated at one end digested with PspXI to produce fragments approximately 33.5 (ii) and 15 kbp (iii) in length (lane 2); and the fragments after isolation and phenol elution from streptavidin beads (lane 3). The red arrow indicates the biotinylated ~15 kbp fragment, (c) Normalized SS-nanopore event histograms of ECD for the initial admixture (top, n = 1158) and the product of bead isolation (bottom, n = 519). SS-nanopore diameters are 6.1 and 6.1 nm, respectively. Lower ECD corresponds to lower molecular weight (i.e., the 15 kbp biotinylated DNA). Insets show a typical conductance trace for each measurement, with initial (open pore) conductance to the left and after addition of the DNA to the right. Spikes indicate molecular translocations. Scale bars are 500 ms (horizontal) and 1 nS (vertical).

    Article Snippet: For bead capture, 500 ng of the resulting mixture was incubated with 25 μ L of streptavidin beads resuspended in 2× binding/washing buffer, with the rest reserved for solid-state (SS)-nanopore measurements.

    Techniques: Isolation, Incubation, Magnetic Beads, Molecular Weight

    SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the streptavidin beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value

    Journal: Nature Communications

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria

    doi: 10.1038/s41467-018-05997-6

    Figure Lengend Snippet: SMRT-Cappable-seq identifies full-length transcripts in bacteria. a Schema of the SMRT-Cappable-seq methodology. 5′ triphosphorylated transcripts are capped with a desthio-biotinylated (DTB) cap analog and bound to the streptavidin beads to specifically capture primary transcripts starting at TSS. The polyadenylation step (A-tailing) ensures the priming of the anchored poly dT primer for cDNA synthesis at the most 3′end of the transcript. b Integrative Genomics Viewer (IGV) representation of the mapping of SMRT-Cappable-seq reads (top) compared to Illumina RNA-seq reads (bottom) in the mprA locus. Forward oriented reads are labeled in pink, reverse oriented reads are labeled in blue. c Comparison between gene expression level in Read counts Per Kilobase of transcript, per Million mapped reads (RPKM) for Illumina RNA-seq and SMRT-Cappable-seq. The Spearman’s rank correlation is 0.798 ( p value

    Article Snippet: The capped RNA was enriched using hydrophilic streptavidin magnetic beads (New England Biolabs).

    Techniques: RNA Sequencing Assay, Labeling, Expressing

    Physical file separations in DENSE storage rescue the decreased sequencing efficiency experienced by high-capacity databases. a , A library of five files was ordered and analyzed using NGS to confirm an even file distribution. b , File 3 strands were enriched over increasingly higher capacity backgrounds of non-specific DNA strands using 30 cycles of random-access PCR. Random access failed to enrich File 3 to above 50% of the total sample once the background capacity reached 31.1 GB, as measured by quantitative PCR. c , DENSE physically extracts a file (orange) from the database so only its strands are sequenced. A primer functionalized with a chemical handle (yellow diamond) is used to execute one emulsion PCR cycle to create chemically labeled copies of the desired file’s strands. Functionalized magnetic beads (brown) that bind to the chemical handle are added to the sample. The desired file is bound to the bead, and the unbound solution containing the original database is removed and saved for future reuse. The bound file is then eluted from the bead. d , After biotin-streptavidin file extractions, the remaining solution still contained all files while the target files were enriched and physically separated, as measured by next generation sequencing. By mapping sequencing reads to the original file sequences, all targeted data were confirmed recovered. The target file was retained in the supernatant containing the database and was able to be copied and extracted again. File 1 was extracted three sequential times, and File 2 was extracted from the solution remaining after an initial extraction of File 1. e , File extractions using fluorescein, digoxigenin, and polyA(25) as chemical handles also successfully separated target files from the database. f , A large-scale background mimicking diverse data was created using error prone PCR 13 to mutagenize and amplify File 1. g , Random access was compared directly to chemical handle extractions. File 3 strands, with a starting fraction of 0.03% of the total number of strands, were enriched over a high-capacity background equivalent to 5.53 TB of undesired, non-specific strands using either random access (black) or PCR followed by chemical handle primer extractions (blue, green, purple or pink). After 5, 15, and 30 cycles of PCR (random access), enrichment of File 3 was 0.0%, 0.0%, and 1.69% of the total sample, respectively. After biotin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 87.5%, and 100% of the total sample, respectively. After fluorescein-modified PCR followed by extraction, the enrichment of File 3 was 0.1%, 49.6%, and 100% of the total sample, respectively. After digoxigenin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 14.2%, and 100% of the total sample, respectively. After poly(A)-25-modified PCR followed by extraction, the enrichment of File 3 was 0.09%, 0.47%, and 100% of the total sample, respectively.

    Journal: bioRxiv

    Article Title: Driving the scalability of DNA-based information storage systems

    doi: 10.1101/591594

    Figure Lengend Snippet: Physical file separations in DENSE storage rescue the decreased sequencing efficiency experienced by high-capacity databases. a , A library of five files was ordered and analyzed using NGS to confirm an even file distribution. b , File 3 strands were enriched over increasingly higher capacity backgrounds of non-specific DNA strands using 30 cycles of random-access PCR. Random access failed to enrich File 3 to above 50% of the total sample once the background capacity reached 31.1 GB, as measured by quantitative PCR. c , DENSE physically extracts a file (orange) from the database so only its strands are sequenced. A primer functionalized with a chemical handle (yellow diamond) is used to execute one emulsion PCR cycle to create chemically labeled copies of the desired file’s strands. Functionalized magnetic beads (brown) that bind to the chemical handle are added to the sample. The desired file is bound to the bead, and the unbound solution containing the original database is removed and saved for future reuse. The bound file is then eluted from the bead. d , After biotin-streptavidin file extractions, the remaining solution still contained all files while the target files were enriched and physically separated, as measured by next generation sequencing. By mapping sequencing reads to the original file sequences, all targeted data were confirmed recovered. The target file was retained in the supernatant containing the database and was able to be copied and extracted again. File 1 was extracted three sequential times, and File 2 was extracted from the solution remaining after an initial extraction of File 1. e , File extractions using fluorescein, digoxigenin, and polyA(25) as chemical handles also successfully separated target files from the database. f , A large-scale background mimicking diverse data was created using error prone PCR 13 to mutagenize and amplify File 1. g , Random access was compared directly to chemical handle extractions. File 3 strands, with a starting fraction of 0.03% of the total number of strands, were enriched over a high-capacity background equivalent to 5.53 TB of undesired, non-specific strands using either random access (black) or PCR followed by chemical handle primer extractions (blue, green, purple or pink). After 5, 15, and 30 cycles of PCR (random access), enrichment of File 3 was 0.0%, 0.0%, and 1.69% of the total sample, respectively. After biotin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 87.5%, and 100% of the total sample, respectively. After fluorescein-modified PCR followed by extraction, the enrichment of File 3 was 0.1%, 49.6%, and 100% of the total sample, respectively. After digoxigenin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 14.2%, and 100% of the total sample, respectively. After poly(A)-25-modified PCR followed by extraction, the enrichment of File 3 was 0.09%, 0.47%, and 100% of the total sample, respectively.

    Article Snippet: PCR amplified samples were purified (AMPure XP beads) and added to prewashed streptavidin magnetic beads (NEB #S1420S) (wash and bind buffer: 20 mM Tris-HCl pH 7.4, 2M NaCl, 2 mM EDTA pH 8) and incubated at room temperature on a rotisserie for 30 minutes.

    Techniques: Sequencing, Next-Generation Sequencing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Labeling, Magnetic Beads, Modification