streptavidin coated magnetic beads  (Thermo Fisher)


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    Name:
    Pierce Streptavidin Magnetic Beads
    Description:
    Thermo Scientific Pierce Streptavidin Magnetic Beads accelerate throughput for automated magnetic purification of biotinylated molecules Features of Streptavidin Magnetic Beads • High performance beads non aggregating pre blocked iron oxide superparamagnetic microparticles provide exceptional uniformity for automated HTS and manual applications alike • Stable immobilization chemistry streptavidin is immobilized using leach resistant chemistry • High capacity superior quality beads with high binding capacity provide rapid and efficient biomolecule purification from complex samples • Low non specific binding stable pre blocked beads provide clean purification products e g antigen eluted in IP with biotinylated antibody that are compatible with mass spectrometry analysis and other downstream analyses • Superior performance nearly three times higher binding capacity than typical beads from other suppliers allowing the use of smaller amounts per experiment Applications • Immunoprecipitate antigens using biotinylated antibodies from a wide variety of sources • Co immunoprecipitate interaction complexes using biotinylated antibodies • Capture protein protein interactions in pull down assays using biotinylated bait proteins • Isolate biotin labeled DNA protein complexes from cell or tissue extracts • Capture single stranded biotinylated DNA oligos • Isolate biotinylated PCR products These streptavidin magnetic beads are validated and optimized for use with high throughput magnetic platforms such as the Thermo Scientific KingFisher 96 and KingFisher Flex Instruments but the beads also enable premium performance for simple benchtop applications using an appropriate magnetic stand The iron oxide super paramagnetic particles offer superior performance high capacity and low nonspecific binding compared with other commercial magnetic beads Pierce Streptavidin Magnetic Beads use a recombinant form of streptavidin with a mass of 53kDa and a near neutral isoelectric point pI The protein is a tetramer having four biotin binding sites Unlike avidin streptavidin has no carbohydrate groups resulting in low nonspecific binding The high affinity interaction between streptavidin and biotin cannot be dissociated efficiently except with very harsh conditions such as boiling in sample loading buffer for SDS PAGE or 8M guanidine HCl pH 1 5 Consequently it is often possible to elute binding partners in an interaction complex without also eluting the biotinylated component
    Catalog Number:
    88816
    Price:
    None
    Category:
    Beads Microspheres
    Applications:
    Magnetic Bead Protein Purification|Protein Biology|Protein Purification|Protein Purification & Isolation
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    Structured Review

    Thermo Fisher streptavidin coated magnetic beads
    Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent <t>Streptavidin)</t> targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.
    Thermo Scientific Pierce Streptavidin Magnetic Beads accelerate throughput for automated magnetic purification of biotinylated molecules Features of Streptavidin Magnetic Beads • High performance beads non aggregating pre blocked iron oxide superparamagnetic microparticles provide exceptional uniformity for automated HTS and manual applications alike • Stable immobilization chemistry streptavidin is immobilized using leach resistant chemistry • High capacity superior quality beads with high binding capacity provide rapid and efficient biomolecule purification from complex samples • Low non specific binding stable pre blocked beads provide clean purification products e g antigen eluted in IP with biotinylated antibody that are compatible with mass spectrometry analysis and other downstream analyses • Superior performance nearly three times higher binding capacity than typical beads from other suppliers allowing the use of smaller amounts per experiment Applications • Immunoprecipitate antigens using biotinylated antibodies from a wide variety of sources • Co immunoprecipitate interaction complexes using biotinylated antibodies • Capture protein protein interactions in pull down assays using biotinylated bait proteins • Isolate biotin labeled DNA protein complexes from cell or tissue extracts • Capture single stranded biotinylated DNA oligos • Isolate biotinylated PCR products These streptavidin magnetic beads are validated and optimized for use with high throughput magnetic platforms such as the Thermo Scientific KingFisher 96 and KingFisher Flex Instruments but the beads also enable premium performance for simple benchtop applications using an appropriate magnetic stand The iron oxide super paramagnetic particles offer superior performance high capacity and low nonspecific binding compared with other commercial magnetic beads Pierce Streptavidin Magnetic Beads use a recombinant form of streptavidin with a mass of 53kDa and a near neutral isoelectric point pI The protein is a tetramer having four biotin binding sites Unlike avidin streptavidin has no carbohydrate groups resulting in low nonspecific binding The high affinity interaction between streptavidin and biotin cannot be dissociated efficiently except with very harsh conditions such as boiling in sample loading buffer for SDS PAGE or 8M guanidine HCl pH 1 5 Consequently it is often possible to elute binding partners in an interaction complex without also eluting the biotinylated component
    https://www.bioz.com/result/streptavidin coated magnetic beads/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    streptavidin coated magnetic beads - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Integration of sample preparation and analysis on an optofluidic chip for multi-target disease detection"

    Article Title: Integration of sample preparation and analysis on an optofluidic chip for multi-target disease detection

    Journal: Lab on a chip

    doi: 10.1039/c8lc00966j

    Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent Streptavidin) targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.
    Figure Legend Snippet: Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent Streptavidin) targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.

    Techniques Used: Acid Assay

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

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

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.02.012

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

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

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

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

    Journal: Gene Expression

    doi:

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

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

    4) Product Images from "Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells"

    Article Title: Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

    Journal: bioRxiv

    doi: 10.1101/736207

    RNA degradation rate between PD-MK and Std conditions did not show significant difference. (A) Schematic representation of the experiment for examining RNA degradation rate. WT mESCs were transiently treated with 4-thiouridine (4sU). By this transient treatment, 4sUs were incorporated into newly synthesized RNA. RNA was then extracted from the cells, and a known amount of spike-in RNA that does not contain 4sU was added. Thereafter, the RNA mix was biotinylated. Then, from a part of this biotinylated RNA mix, biotinylated RNA was removed using streptavidin beads, and unbiotinylated RNAs that were transcribed before the addition of 4sU were recovered. RNA samples that were not treated with streptavidin beads contained both existing RNA and newly synthesized RNA. Therefore, we refer to these RNA samples as total RNAs. These were reverse transcribed and analyzed by qPCR. (B) A bar graph of the ratio of unbound and total RNA. For many samples, the ratio has exceeded 1, which was theoretically impossible. It is considered that the reverse transcription efficiency of 4sU-introduced RNA could be extremely low (see Material and Methods). (C) We assumed that the presence of biotinylated RNA during reverse transcription may trap reverse transcriptase, and that the efficiency of reverse transcription is further reduced globally. We assume that the global suppression effect of reverse transcriptase trapping is I g (global inhibitory effect). Moreover, the reverse transcription inhibitory effect of biotinylated RNA itself was defined as I s (see Material and Methods). In order to determine the appropriate value of I s , several values were assigned to I s , and mRNA half-lives in the Std condition were compared with the previously reported mRNA half-lives ( 21 ). We found that the scaling of mRNA half-lives in the Std condition and that of previously reported mRNA half-lives were getting closer when I s was 0.1. (D) Average mRNA half-life. The half-life of mRNA was calculated using the ratio obtained in (B) and the correction formula (see Material and Methods). In all genes analyzed, there was no significant difference between PD-MK and Std conditions.
    Figure Legend Snippet: RNA degradation rate between PD-MK and Std conditions did not show significant difference. (A) Schematic representation of the experiment for examining RNA degradation rate. WT mESCs were transiently treated with 4-thiouridine (4sU). By this transient treatment, 4sUs were incorporated into newly synthesized RNA. RNA was then extracted from the cells, and a known amount of spike-in RNA that does not contain 4sU was added. Thereafter, the RNA mix was biotinylated. Then, from a part of this biotinylated RNA mix, biotinylated RNA was removed using streptavidin beads, and unbiotinylated RNAs that were transcribed before the addition of 4sU were recovered. RNA samples that were not treated with streptavidin beads contained both existing RNA and newly synthesized RNA. Therefore, we refer to these RNA samples as total RNAs. These were reverse transcribed and analyzed by qPCR. (B) A bar graph of the ratio of unbound and total RNA. For many samples, the ratio has exceeded 1, which was theoretically impossible. It is considered that the reverse transcription efficiency of 4sU-introduced RNA could be extremely low (see Material and Methods). (C) We assumed that the presence of biotinylated RNA during reverse transcription may trap reverse transcriptase, and that the efficiency of reverse transcription is further reduced globally. We assume that the global suppression effect of reverse transcriptase trapping is I g (global inhibitory effect). Moreover, the reverse transcription inhibitory effect of biotinylated RNA itself was defined as I s (see Material and Methods). In order to determine the appropriate value of I s , several values were assigned to I s , and mRNA half-lives in the Std condition were compared with the previously reported mRNA half-lives ( 21 ). We found that the scaling of mRNA half-lives in the Std condition and that of previously reported mRNA half-lives were getting closer when I s was 0.1. (D) Average mRNA half-life. The half-life of mRNA was calculated using the ratio obtained in (B) and the correction formula (see Material and Methods). In all genes analyzed, there was no significant difference between PD-MK and Std conditions.

    Techniques Used: Synthesized, Real-time Polymerase Chain Reaction

    5) Product Images from "Development of a dual aptamer-based multiplex protein biosensor"

    Article Title: Development of a dual aptamer-based multiplex protein biosensor

    Journal: Biosensors & bioelectronics

    doi: 10.1016/j.bios.2010.04.034

    Dual-aptamer detection schematic Thrombin and PDGF-BB are represented by triangles and double-ellipses, respectively. The sensing aptamers (MB-T, MB-P) containing thrombin aptamer (Apt 1A) and PDGF-BB aptamer (Apt 2A) are each labeled with an MB tag (MB1 and MB2) and flanked by a pair of universal primers (5′p and 3′p). Capture aptamers (Bio-T and Bio-P) containing thrombin aptamer (Apt 1B) and PDGF-BB aptamer (Apt 2B) are biotinylated to be attached to streptavidin-coated magnetic beads. Oligo(dT)s are used as the spacer between the aptamer sequences and the beads. After separation, eluted sensing aptamers are amplified by multiplex PCR and quantified.
    Figure Legend Snippet: Dual-aptamer detection schematic Thrombin and PDGF-BB are represented by triangles and double-ellipses, respectively. The sensing aptamers (MB-T, MB-P) containing thrombin aptamer (Apt 1A) and PDGF-BB aptamer (Apt 2A) are each labeled with an MB tag (MB1 and MB2) and flanked by a pair of universal primers (5′p and 3′p). Capture aptamers (Bio-T and Bio-P) containing thrombin aptamer (Apt 1B) and PDGF-BB aptamer (Apt 2B) are biotinylated to be attached to streptavidin-coated magnetic beads. Oligo(dT)s are used as the spacer between the aptamer sequences and the beads. After separation, eluted sensing aptamers are amplified by multiplex PCR and quantified.

    Techniques Used: Labeling, Magnetic Beads, Amplification, Multiplex Assay, Polymerase Chain Reaction

    6) Product Images from "Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities"

    Article Title: Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities

    Journal: Nature Communications

    doi: 10.1038/ncomms8581

    Active translocating Upf1 disrupts protein–NA interactions. ( a ) Time course for streptavidin displacement from 3′-biotinylated RNA by Upf1-HD translocation. Data points ( Supplementary Fig. S5a ) were fitted with y = A (1− e –kt ) 57 (error bars are s.d.) ( b ) Force-jump experiment showing that Gp32-B (300 nM) covers ssDNA. In the absence of Upf1, when the hairpin is open, Gp32-B binds DNA, as seen by the slowdown of the DNA hairpin refolding (from 4,080 s to about 4,082 s, F =8 pN, refolding rate of 1,000 bp s −1 ). Gp32-B is known to efficiently bind ssDNA with a dissociation rate measured as ∼1 molecule per second on 65 bp s −1 (refs 58 , 59 ). ( c ) Single-molecule analysis of Gp32-B displacement from ssDNA by translocating Upf1-HD (from 2,840 to 3,480 s) The Gp32-B protein was stripped off from ssDNA in presence of Upf1-HD and ATP (right panel). During Upf1 translocation, Gp32-B attached on the displaced strand may transiently block the fork refolding as seen in the time window (from 3,080 to 3,270 s).
    Figure Legend Snippet: Active translocating Upf1 disrupts protein–NA interactions. ( a ) Time course for streptavidin displacement from 3′-biotinylated RNA by Upf1-HD translocation. Data points ( Supplementary Fig. S5a ) were fitted with y = A (1− e –kt ) 57 (error bars are s.d.) ( b ) Force-jump experiment showing that Gp32-B (300 nM) covers ssDNA. In the absence of Upf1, when the hairpin is open, Gp32-B binds DNA, as seen by the slowdown of the DNA hairpin refolding (from 4,080 s to about 4,082 s, F =8 pN, refolding rate of 1,000 bp s −1 ). Gp32-B is known to efficiently bind ssDNA with a dissociation rate measured as ∼1 molecule per second on 65 bp s −1 (refs 58 , 59 ). ( c ) Single-molecule analysis of Gp32-B displacement from ssDNA by translocating Upf1-HD (from 2,840 to 3,480 s) The Gp32-B protein was stripped off from ssDNA in presence of Upf1-HD and ATP (right panel). During Upf1 translocation, Gp32-B attached on the displaced strand may transiently block the fork refolding as seen in the time window (from 3,080 to 3,270 s).

    Techniques Used: Translocation Assay, Blocking Assay

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep12090

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

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

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

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

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1468

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

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

    9) Product Images from "Selection of aptamers for a protein target in cell lysate and their application to protein purification"

    Article Title: Selection of aptamers for a protein target in cell lysate and their application to protein purification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp176

    SDS–PAGE with Coomassie staining of affinity pull-down of MutS from the E. coli cell lysate using the aptamers developed by AptaPIC. The lanes from left to right correspond to: (i) molecular weight standards (×1000); (ii) pure MutS; (iii) MutS-containing cell lysate, the sample contained 91.25 µg/ml and 18 µg/ml of MutS; (iv) MutS purification using the naive DNA library, bound fraction; (v) MutS purification using the developed aptamer pool, bound fraction; (vi) fraction bound to streptavidin-coated magnetic beads; (vii) MutS purification using the naive DNA library, unbound fraction; and (viii) MutS purification using the developed aptamer pool, unbound fraction.
    Figure Legend Snippet: SDS–PAGE with Coomassie staining of affinity pull-down of MutS from the E. coli cell lysate using the aptamers developed by AptaPIC. The lanes from left to right correspond to: (i) molecular weight standards (×1000); (ii) pure MutS; (iii) MutS-containing cell lysate, the sample contained 91.25 µg/ml and 18 µg/ml of MutS; (iv) MutS purification using the naive DNA library, bound fraction; (v) MutS purification using the developed aptamer pool, bound fraction; (vi) fraction bound to streptavidin-coated magnetic beads; (vii) MutS purification using the naive DNA library, unbound fraction; and (viii) MutS purification using the developed aptamer pool, unbound fraction.

    Techniques Used: SDS Page, Staining, Molecular Weight, Purification, Magnetic Beads

    10) Product Images from "Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models"

    Article Title: Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01691

    Development of a classical pathway C3 and C5 convertase model. (A) In the CP convertase model, streptavidin beads are labeled with biotinylated DNP which serves as a model antigen. Addition of IgG1 recognizing DNP and complement proteins results in formation of C4b2a and C4b2a(C3b) n convertases on the bead surface, which convert C3 and C5. (B) Only in the presence of all CP components (antigen, IgG, and CP proteins) C3 and C5 are converted, as measured by calcium mobilization in U937-C3aR and U937-C5aR cells, respectively. (C) C3 conversion via CP convertases results in release of C3a in the supernatant (as shown by calcium mobilization) and (D) C3b deposition on the bead surface (as shown by flow cytometry). Identical amounts of the non-reactive C3 variants C3b H2O and C3 MA do not bind to the bead surface. (E) C5 convertase activity increases with the level of deposited C3b molecules on the beads surface, but is not affected by C3b H2O or C3 MA in solution. Uncoupling C3 and C5 conversion by introduction of an extra washing step does not alter C5 conversion, indicating that CP C5 conversion only depends on deposited C3b molecules around existing C3 convertases. (B–E) Data of three independent experiments, presented as mean ± SD.
    Figure Legend Snippet: Development of a classical pathway C3 and C5 convertase model. (A) In the CP convertase model, streptavidin beads are labeled with biotinylated DNP which serves as a model antigen. Addition of IgG1 recognizing DNP and complement proteins results in formation of C4b2a and C4b2a(C3b) n convertases on the bead surface, which convert C3 and C5. (B) Only in the presence of all CP components (antigen, IgG, and CP proteins) C3 and C5 are converted, as measured by calcium mobilization in U937-C3aR and U937-C5aR cells, respectively. (C) C3 conversion via CP convertases results in release of C3a in the supernatant (as shown by calcium mobilization) and (D) C3b deposition on the bead surface (as shown by flow cytometry). Identical amounts of the non-reactive C3 variants C3b H2O and C3 MA do not bind to the bead surface. (E) C5 convertase activity increases with the level of deposited C3b molecules on the beads surface, but is not affected by C3b H2O or C3 MA in solution. Uncoupling C3 and C5 conversion by introduction of an extra washing step does not alter C5 conversion, indicating that CP C5 conversion only depends on deposited C3b molecules around existing C3 convertases. (B–E) Data of three independent experiments, presented as mean ± SD.

    Techniques Used: Labeling, Flow Cytometry, Cytometry, Activity Assay

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008074

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

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

    12) Product Images from "Streptomyces Telomeres Contain a Promoter ▿"

    Article Title: Streptomyces Telomeres Contain a Promoter ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01299-08

    Isolation of TBPs. Biotin-labeled double-stranded (DS167) and single-stranded (SS167w and SS167c) DNA fragments immobilized in a streptavidin column were used to purify binding proteins in crude extracts of S. lividans . The eluted proteins were separated by electrophoresis in SDS-polyacrylamide gel and silver stained (left). The sizes (in kDa) of the most abundant TBPs are indicated. The asterisk depicts nonspecific binding proteins that were also present in the flowthrough fractions. The region containing the β and β′ subunits (bb′) of RNA polymerase is enlarged in the insert on the left. The separated proteins were analyzed by immunoblots using anti-α (anti-a, middle) and anti-HrdB (right) antibody.
    Figure Legend Snippet: Isolation of TBPs. Biotin-labeled double-stranded (DS167) and single-stranded (SS167w and SS167c) DNA fragments immobilized in a streptavidin column were used to purify binding proteins in crude extracts of S. lividans . The eluted proteins were separated by electrophoresis in SDS-polyacrylamide gel and silver stained (left). The sizes (in kDa) of the most abundant TBPs are indicated. The asterisk depicts nonspecific binding proteins that were also present in the flowthrough fractions. The region containing the β and β′ subunits (bb′) of RNA polymerase is enlarged in the insert on the left. The separated proteins were analyzed by immunoblots using anti-α (anti-a, middle) and anti-HrdB (right) antibody.

    Techniques Used: Isolation, Labeling, Binding Assay, Electrophoresis, Staining, Western Blot

    Design and synthesis of telomere DNA probes. (A) The sequence and predicted secondary structure of the 3′ ends of the S. lividans ). The first 167 nt spanning the first seven palindromes is framed. The 12 substitutions (in lightface) in the corresponding terminal sequence of the S. coelicolor chromosome are shown next to the corresponding residues. Inset, secondary structure of the coliphage N4 promoter; M, A or G. (B) Synthesis of double-stranded and single-stranded terminal DNA. A pair of primers (Forward and Reverse), one of which was end labeled with biotin (filled circles), was used to produce the DS167 DNA by PCR. The two biotin-labeled strands (SS167w and SS167c) were separately purified by denaturation of DS167 DNA, followed by binding to streptavidin-coated magnetic beads.
    Figure Legend Snippet: Design and synthesis of telomere DNA probes. (A) The sequence and predicted secondary structure of the 3′ ends of the S. lividans ). The first 167 nt spanning the first seven palindromes is framed. The 12 substitutions (in lightface) in the corresponding terminal sequence of the S. coelicolor chromosome are shown next to the corresponding residues. Inset, secondary structure of the coliphage N4 promoter; M, A or G. (B) Synthesis of double-stranded and single-stranded terminal DNA. A pair of primers (Forward and Reverse), one of which was end labeled with biotin (filled circles), was used to produce the DS167 DNA by PCR. The two biotin-labeled strands (SS167w and SS167c) were separately purified by denaturation of DS167 DNA, followed by binding to streptavidin-coated magnetic beads.

    Techniques Used: Sequencing, Labeling, Polymerase Chain Reaction, Purification, Binding Assay, Magnetic Beads

    13) Product Images from "Integration of sample preparation and analysis on an optofluidic chip for multi-target disease detection"

    Article Title: Integration of sample preparation and analysis on an optofluidic chip for multi-target disease detection

    Journal: Lab on a chip

    doi: 10.1039/c8lc00966j

    Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent Streptavidin) targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.
    Figure Legend Snippet: Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent Streptavidin) targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.

    Techniques Used: Acid Assay

    14) Product Images from "MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.01613-18

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

    Techniques Used: Transfection, Western Blot, Labeling

    15) Product Images from "Measuring DNA mechanics on the genome scale"

    Article Title: Measuring DNA mechanics on the genome scale

    Journal: Nature

    doi: 10.1038/s41586-020-03052-3

    Dependence of cyclizability on tether geometry and rotational phasing. a, ). Plotted is the mean A/T content as a function of position along the central variable 50 bp region, where the mean is calculated by averaging over the 1,000 most cyclizable (red) or least cyclizable (blue) sequences. The value of n (n = 26) is the distance in nucleotides of the biotin tether from the end of each molecule in the library (). b, In an untethered geometry, sequence features such as the phase of the oscillations in A/T content may result in the looped configuration having a preference for a certain poloidal angle (rotation along the long axis of DNA). Preference for a certain poloidal angle translates to a preference for a certain orientation of the biotin-streptavidin tether. Shown above are two extreme cases – in one case, the poloidal angle preference of the sequence results in a preferential orientation of the tether on the outside, while in the other case, the tether points to the inside at the point of contact with the DNA. As the biotin-streptavidin-complex is quite large, the outside orientation may be more favored for looping owing to steric considerations. The outside orientation can be converted to the inside orientation by moving the biotin tether point to a base that is half the DNA helical repeat away. This may explain why the phase of the oscillation of A/T content among the most or least cyclizable sequences shifts by half the helical repeat of DNA when the tether point is also shifted by half the helical repeat of DNA (about 5 bases) (compare panels a and c). c, The random library was re-prepared, placing the biotin 31 nt away from the ends (n = 31) and loop-seq was performed. Plotted are the same quantities as in panel a, except the 1,000 most and least cyclizable sequences of the library were identified based on the newly obtained cyclizability values under the n = 31 condition. d – e, . In panel e, Pearson’s r = 0.987, 95% CI = [0.986, 0.987]. p
    Figure Legend Snippet: Dependence of cyclizability on tether geometry and rotational phasing. a, ). Plotted is the mean A/T content as a function of position along the central variable 50 bp region, where the mean is calculated by averaging over the 1,000 most cyclizable (red) or least cyclizable (blue) sequences. The value of n (n = 26) is the distance in nucleotides of the biotin tether from the end of each molecule in the library (). b, In an untethered geometry, sequence features such as the phase of the oscillations in A/T content may result in the looped configuration having a preference for a certain poloidal angle (rotation along the long axis of DNA). Preference for a certain poloidal angle translates to a preference for a certain orientation of the biotin-streptavidin tether. Shown above are two extreme cases – in one case, the poloidal angle preference of the sequence results in a preferential orientation of the tether on the outside, while in the other case, the tether points to the inside at the point of contact with the DNA. As the biotin-streptavidin-complex is quite large, the outside orientation may be more favored for looping owing to steric considerations. The outside orientation can be converted to the inside orientation by moving the biotin tether point to a base that is half the DNA helical repeat away. This may explain why the phase of the oscillation of A/T content among the most or least cyclizable sequences shifts by half the helical repeat of DNA when the tether point is also shifted by half the helical repeat of DNA (about 5 bases) (compare panels a and c). c, The random library was re-prepared, placing the biotin 31 nt away from the ends (n = 31) and loop-seq was performed. Plotted are the same quantities as in panel a, except the 1,000 most and least cyclizable sequences of the library were identified based on the newly obtained cyclizability values under the n = 31 condition. d – e, . In panel e, Pearson’s r = 0.987, 95% CI = [0.986, 0.987]. p

    Techniques Used: Sequencing

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

    Article Title: Histone modifications influence mediator interactions with chromatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr551

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

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

    17) Product Images from "Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification"

    Article Title: Droplet Digital Enzyme-Linked Oligonucleotide Hybridization Assay for Absolute RNA Quantification

    Journal: Scientific Reports

    doi: 10.1038/srep13795

    Continuous flow droplet digital ELOHA for absolute RNA quantification based on two dependent Poisson process. ( a ) Schematic of droplet digital ELOHA on a microfluidic chip. Initially, a sandwiched complex (inset i, first Poisson process) is formed by hybridization of oligos on magnetic beads. The enzyme labels (SβG) are then attached to the sandwiched complex through biotin-streptavidin interaction. This magnetic bead suspension and the fluorogenic substrate (RGP), each of a volume of 50 μL, are loaded into separate capillary tubes. Droplets are continuously generated by shearing the bead/substrate mixture with oil/surfactant on the device (inset ii, second dependent Poisson process). After continuous incubation of the droplets at room temperature in-line, the fluorescence intensity of each droplet is recorded one by one through a custom designed optical system (inset iii). ( b ) Schematic of custom designed optical system. The system includes a trans-illumination source for imaging the droplets on the chip, and two laser sources for fluorescence excitation. Abbreviation: DM, dichroic mirror; BP: band pass; APD: avalanche photodiode; CCD: CCD Camera.
    Figure Legend Snippet: Continuous flow droplet digital ELOHA for absolute RNA quantification based on two dependent Poisson process. ( a ) Schematic of droplet digital ELOHA on a microfluidic chip. Initially, a sandwiched complex (inset i, first Poisson process) is formed by hybridization of oligos on magnetic beads. The enzyme labels (SβG) are then attached to the sandwiched complex through biotin-streptavidin interaction. This magnetic bead suspension and the fluorogenic substrate (RGP), each of a volume of 50 μL, are loaded into separate capillary tubes. Droplets are continuously generated by shearing the bead/substrate mixture with oil/surfactant on the device (inset ii, second dependent Poisson process). After continuous incubation of the droplets at room temperature in-line, the fluorescence intensity of each droplet is recorded one by one through a custom designed optical system (inset iii). ( b ) Schematic of custom designed optical system. The system includes a trans-illumination source for imaging the droplets on the chip, and two laser sources for fluorescence excitation. Abbreviation: DM, dichroic mirror; BP: band pass; APD: avalanche photodiode; CCD: CCD Camera.

    Techniques Used: Flow Cytometry, Chromatin Immunoprecipitation, Hybridization, Magnetic Beads, Generated, Incubation, Fluorescence, Imaging

    18) Product Images from "Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins"

    Article Title: Mapping the Proteome of the Synaptic Cleft through Proximity Labeling Reveals New Cleft Proteins

    Journal: Proteomes

    doi: 10.3390/proteomes6040048

    SynCAM 1-peroxidase fusion protein peroxidase-mediated proximity labeling in the synaptic cleft. ( A ]) peroxidase was inserted at the base of the SynCAM 1 extracellular domain, with immunoglobulin (Ig) domains, trans-membrane (TM) region, and intracellular PDZ domain interaction sequence indicated. APEX2 or HRP catalyzes the formation of a short-lived biotin-AEEA-phenoxyl radical (red dot) after exogenous addition of H 2 O 2 and membrane-impermeable biotin-AEEA-phenol (blue dot). ( B ) Exogenous biotin-AEEA-phenol induced biotinylation only at the cell surface. Staining for biotin (visualized by StreptAvidin-Alexa488) in HEK293T cells expressing SynCAM 1-APEX2 in presence (+) but not in absence (−) of H 2 O 2 . ( C ) Exogenous biotin-AEEA-phenol did not induce biotinylation in HEK293T cells expressing cytosolic APEX2-NES.
    Figure Legend Snippet: SynCAM 1-peroxidase fusion protein peroxidase-mediated proximity labeling in the synaptic cleft. ( A ]) peroxidase was inserted at the base of the SynCAM 1 extracellular domain, with immunoglobulin (Ig) domains, trans-membrane (TM) region, and intracellular PDZ domain interaction sequence indicated. APEX2 or HRP catalyzes the formation of a short-lived biotin-AEEA-phenoxyl radical (red dot) after exogenous addition of H 2 O 2 and membrane-impermeable biotin-AEEA-phenol (blue dot). ( B ) Exogenous biotin-AEEA-phenol induced biotinylation only at the cell surface. Staining for biotin (visualized by StreptAvidin-Alexa488) in HEK293T cells expressing SynCAM 1-APEX2 in presence (+) but not in absence (−) of H 2 O 2 . ( C ) Exogenous biotin-AEEA-phenol did not induce biotinylation in HEK293T cells expressing cytosolic APEX2-NES.

    Techniques Used: Labeling, Sequencing, Staining, Expressing

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149774

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

    Techniques Used: Magnetic Beads, Blocking Assay

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

    Techniques Used: Binding Assay, Magnetic Beads, Ligation

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

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

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00259

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

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

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep04069

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

    Techniques Used: Cell Culture, Incubation, Labeling

    22) Product Images from "Split-TurboID enables contact-dependent proximity labeling in cells"

    Article Title: Split-TurboID enables contact-dependent proximity labeling in cells

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.988022

    Characterization of low-affinity split-TurboID. (A) Confocal fluorescence imaging of low-affinity split-TurboID (split site L73/G74). HEK293T cells were transiently transfected and incubated with 50 μM biotin and 100 nM rapamycin for 1 hour, then fixed and stained with anti-V5 to detect the N-terminal fragment (Tb(N)), anti-HA to detect the C-terminal fragment (Tb(C)), and neutravidin-647 to detect biotinylated proteins. Scale bars, 20 μm. (B) Split-TurboID time course. HEK293T cells transiently transfected with split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for the indicated times, and whole cell lysates were analyzed by streptavidin blotting. Full-length TurboID (10 minutes) and full-length BioID (1 hour and 18 hours) were included for comparison. (C) Design of constructs used to target split-TurboID fragments to various cellular compartments. (MTS = mitochondrial targeting sequence, SS = signal sequence) (D) Confocal fluorescence imaging of low-affinity split-TurboID targeted to various cellular compartments. HEK293T cells were labeled and imaged as in (A). Fluorescence intensities are not normalized across cellular compartments. Zoomed images of the boxed regions are included. Scale bars, 20 μm.
    Figure Legend Snippet: Characterization of low-affinity split-TurboID. (A) Confocal fluorescence imaging of low-affinity split-TurboID (split site L73/G74). HEK293T cells were transiently transfected and incubated with 50 μM biotin and 100 nM rapamycin for 1 hour, then fixed and stained with anti-V5 to detect the N-terminal fragment (Tb(N)), anti-HA to detect the C-terminal fragment (Tb(C)), and neutravidin-647 to detect biotinylated proteins. Scale bars, 20 μm. (B) Split-TurboID time course. HEK293T cells transiently transfected with split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for the indicated times, and whole cell lysates were analyzed by streptavidin blotting. Full-length TurboID (10 minutes) and full-length BioID (1 hour and 18 hours) were included for comparison. (C) Design of constructs used to target split-TurboID fragments to various cellular compartments. (MTS = mitochondrial targeting sequence, SS = signal sequence) (D) Confocal fluorescence imaging of low-affinity split-TurboID targeted to various cellular compartments. HEK293T cells were labeled and imaged as in (A). Fluorescence intensities are not normalized across cellular compartments. Zoomed images of the boxed regions are included. Scale bars, 20 μm.

    Techniques Used: Fluorescence, Imaging, Transfection, Incubation, Staining, Construct, Sequencing, Labeling

    Characterization of stable HEK293T cell lines expressing split-TurboID used for ER-mitochondria proteomics. Related to Figure 3 . (A) Confocal fluorescence imaging of split-TurboID targeted to the OMM and ERM. Cells stably expressing split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 1 hour, then fixed and stained with anti-V5 to detect the OMM-targeted N-terminal fragment (Tb(N)), anti-HA to detect the ERM-targeted C-terminal fragment (Tb(C)), and neutravidin-647 to detect biotinylated proteins. Scale bars, 20 μm. (B) Cells stably expressing OMM/ERM-targeted split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 0, 1, or 4 hours. Cells were labeled with 500 nM MitoTracker for 30 minutes then fixed and stained with anti-CANX, which is an ER marker and neutravidin-647 to detect biotinylated proteins. (C) Quantification of colocalization between CANX and MitoTracker in (B) (using a threshold of > 15 times background signal). Quantitation from 5 fields of view per condition are included. (D) Enrichment of biotinylated proteins for proteomics. HEK293T cells stably expressing OMM/ERM-targeted split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 4 hours. HEK293T cells stably expressing NES-, OMM-, or ERM-targeted full-length TurboID were treated with 50 μM biotin for 1 minute. Whole cell lysates were analyzed by streptavidin blotting. Asterisks indicate ligase self-biotinylation. (E) Samples were generated as in (D). Biotinylated proteins were enriched from lysates using streptavidin beads, eluted, and analyzed by streptavidin blotting. Asterisks indicate ligase self-biotinylation
    Figure Legend Snippet: Characterization of stable HEK293T cell lines expressing split-TurboID used for ER-mitochondria proteomics. Related to Figure 3 . (A) Confocal fluorescence imaging of split-TurboID targeted to the OMM and ERM. Cells stably expressing split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 1 hour, then fixed and stained with anti-V5 to detect the OMM-targeted N-terminal fragment (Tb(N)), anti-HA to detect the ERM-targeted C-terminal fragment (Tb(C)), and neutravidin-647 to detect biotinylated proteins. Scale bars, 20 μm. (B) Cells stably expressing OMM/ERM-targeted split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 0, 1, or 4 hours. Cells were labeled with 500 nM MitoTracker for 30 minutes then fixed and stained with anti-CANX, which is an ER marker and neutravidin-647 to detect biotinylated proteins. (C) Quantification of colocalization between CANX and MitoTracker in (B) (using a threshold of > 15 times background signal). Quantitation from 5 fields of view per condition are included. (D) Enrichment of biotinylated proteins for proteomics. HEK293T cells stably expressing OMM/ERM-targeted split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 4 hours. HEK293T cells stably expressing NES-, OMM-, or ERM-targeted full-length TurboID were treated with 50 μM biotin for 1 minute. Whole cell lysates were analyzed by streptavidin blotting. Asterisks indicate ligase self-biotinylation. (E) Samples were generated as in (D). Biotinylated proteins were enriched from lysates using streptavidin beads, eluted, and analyzed by streptavidin blotting. Asterisks indicate ligase self-biotinylation

    Techniques Used: Expressing, Fluorescence, Imaging, Stable Transfection, Construct, Staining, Labeling, Marker, Quantitation Assay, Generated

    Reconstitution of split-TurboID at ER-mitochondria contact sites. (A) Schematic of split-TurboID reconstitution across ER-mitochondria contacts in the presence or absence of rapamycin for inducing dimerization. (B) Design of constructs targeting split-TurboID fragments to the outer mitochondrial membrane (OMM) and ER membrane (ERM). (OMM-targeted construct = 259 amino acids; ERM-targeted construct = 429 amino acids.) (C) Confocal fluorescence imaging of split-TurboID activity at ER-mitochondria contacts. Constructs were introduced into U2OS cells using lentivirus. Two days after transduction, cells were incubated with 50 μM biotin and 100 nM rapamycin for 1 hour, then fixed and stained with anti-V5 to detect the N-terminal fragment (Tb(N)), anti-HA to detect the C-terminal fragment (Tb(C)), and neutravidin-647 to detect biotinylated proteins. Zoomed images of the boxed regions are included. Scale bars, 20 μm. (D) Localization of split-TurboID in HEK293T cells stably expressing constructs from (B). Cells were fixed and stained with anti-V5 to detect the OMM-targeted N-terminal fragment (Tb(N)) or with anti-HA to detect the ERM-targeted C-terminal fragment (Tb(C)). Tom20 and mCherry-KDEL were used as mitochondrial and ER markers, respectively. Scale bars, 10 μm. Colocalization of V5 with Tom20 and HA with mCherry-KDEL are shown on the right. Quantitation from 5 fields of view per condition are included. (E) Enrichment of known ER-mitochondria proteins by split-TurboID-catalyzed proximity labeling. HEK293T cells stably expressing OMM/ERM-targeted split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 4 hours. HEK293T cells stably expressing NES-, OMM-, or ERM-targeted full-length TurboID were treated with 50 μM biotin for 1 minute. Biotinylated proteins were enriched from lysates using streptavidin beads, eluted, and analyzed by streptavidin blotting. Streptavidin-enriched material was probed with antibodies against FACL4 and Mff. Asterisks indicate ligase self-biotinylation. (F) Enrichment of biotinylated proteins for proteomics. Samples were generated as in (E). Biotinylated proteins were enriched from lysates using streptavidin beads, eluted, and analyzed by silver stain.
    Figure Legend Snippet: Reconstitution of split-TurboID at ER-mitochondria contact sites. (A) Schematic of split-TurboID reconstitution across ER-mitochondria contacts in the presence or absence of rapamycin for inducing dimerization. (B) Design of constructs targeting split-TurboID fragments to the outer mitochondrial membrane (OMM) and ER membrane (ERM). (OMM-targeted construct = 259 amino acids; ERM-targeted construct = 429 amino acids.) (C) Confocal fluorescence imaging of split-TurboID activity at ER-mitochondria contacts. Constructs were introduced into U2OS cells using lentivirus. Two days after transduction, cells were incubated with 50 μM biotin and 100 nM rapamycin for 1 hour, then fixed and stained with anti-V5 to detect the N-terminal fragment (Tb(N)), anti-HA to detect the C-terminal fragment (Tb(C)), and neutravidin-647 to detect biotinylated proteins. Zoomed images of the boxed regions are included. Scale bars, 20 μm. (D) Localization of split-TurboID in HEK293T cells stably expressing constructs from (B). Cells were fixed and stained with anti-V5 to detect the OMM-targeted N-terminal fragment (Tb(N)) or with anti-HA to detect the ERM-targeted C-terminal fragment (Tb(C)). Tom20 and mCherry-KDEL were used as mitochondrial and ER markers, respectively. Scale bars, 10 μm. Colocalization of V5 with Tom20 and HA with mCherry-KDEL are shown on the right. Quantitation from 5 fields of view per condition are included. (E) Enrichment of known ER-mitochondria proteins by split-TurboID-catalyzed proximity labeling. HEK293T cells stably expressing OMM/ERM-targeted split-TurboID constructs were treated with 50 μM biotin and 100 nM rapamycin for 4 hours. HEK293T cells stably expressing NES-, OMM-, or ERM-targeted full-length TurboID were treated with 50 μM biotin for 1 minute. Biotinylated proteins were enriched from lysates using streptavidin beads, eluted, and analyzed by streptavidin blotting. Streptavidin-enriched material was probed with antibodies against FACL4 and Mff. Asterisks indicate ligase self-biotinylation. (F) Enrichment of biotinylated proteins for proteomics. Samples were generated as in (E). Biotinylated proteins were enriched from lysates using streptavidin beads, eluted, and analyzed by silver stain.

    Techniques Used: Construct, Fluorescence, Imaging, Activity Assay, Transduction, Incubation, Staining, Stable Transfection, Expressing, Quantitation Assay, Labeling, Generated, Silver Staining

    Screening of TurboID split sites. Related to Figure 1 . (A) Comparison of split sites in HEK293T cells. Cells transiently transfected with the indicated constructs were treated with 50 μM biotin and 100 nM rapamycin for 24 hours, and whole cell lysates were analyzed by streptavidin blotting. For each construct pair, lanes are shown with both fragments present (B), N-terminal fragment only (N), or C-terminal fragment only (C). Anti-V5 and anti-HA staining show expression levels of N- and C-terminal fragments, respectively. Full-length TurboID (30 minutes labeling) and full-length BioID were included for comparison. Dashed lines indicate separate blots performed at the same time and developed simultaneously. Quantification of biotinylation activity is shown in Figure 1C . (B) Testing of additional split sites around the most promising split (73/74) from the first round of screening in HEK293T cells. Cells transiently transfected with the indicated constructs were treated with 50 μM biotin and 100 nM rapamycin for 1 hour, and whole cell lysates were analyzed by streptavidin blotting. For each split site, the N-terminal and C-terminal fragments were either expressed alone (N or C) or both together (B). Anti-V5 and anti-HA staining show expression levels of N- and C-terminal fragments, respectively. Full-length TurboID (treated with 50 μM biotin for 1 hour) and untransfected samples were included for comparison. (C) Testing additional combinations of N-terminal and C-terminal fragments from (C) in HEK293T cells. Samples were analyzed as described in (B), with 1 hour of labeling. Full-length TurboID and BioID treated with 50 μM biotin for 1 hour, full-length BioID treated with 50 μM biotin for 18 hours, and untransfected samples were included for comparison.
    Figure Legend Snippet: Screening of TurboID split sites. Related to Figure 1 . (A) Comparison of split sites in HEK293T cells. Cells transiently transfected with the indicated constructs were treated with 50 μM biotin and 100 nM rapamycin for 24 hours, and whole cell lysates were analyzed by streptavidin blotting. For each construct pair, lanes are shown with both fragments present (B), N-terminal fragment only (N), or C-terminal fragment only (C). Anti-V5 and anti-HA staining show expression levels of N- and C-terminal fragments, respectively. Full-length TurboID (30 minutes labeling) and full-length BioID were included for comparison. Dashed lines indicate separate blots performed at the same time and developed simultaneously. Quantification of biotinylation activity is shown in Figure 1C . (B) Testing of additional split sites around the most promising split (73/74) from the first round of screening in HEK293T cells. Cells transiently transfected with the indicated constructs were treated with 50 μM biotin and 100 nM rapamycin for 1 hour, and whole cell lysates were analyzed by streptavidin blotting. For each split site, the N-terminal and C-terminal fragments were either expressed alone (N or C) or both together (B). Anti-V5 and anti-HA staining show expression levels of N- and C-terminal fragments, respectively. Full-length TurboID (treated with 50 μM biotin for 1 hour) and untransfected samples were included for comparison. (C) Testing additional combinations of N-terminal and C-terminal fragments from (C) in HEK293T cells. Samples were analyzed as described in (B), with 1 hour of labeling. Full-length TurboID and BioID treated with 50 μM biotin for 1 hour, full-length BioID treated with 50 μM biotin for 18 hours, and untransfected samples were included for comparison.

    Techniques Used: Transfection, Construct, Staining, Expressing, Labeling, Activity Assay

    Proteomic mapping of ER-mitochondria contacts in HEK293T cells. (A) Experimental design and labeling conditions for MS-based proteomics. Cells stably expressing the indicated constructs were labeled with 50 μM biotin and 100 nM rapamycin. Split-TurboID (ERM/OMM) samples were labeled for 4 hours and full-length TurboID samples were labeled for 1 minute. Cells were then lysed, and biotinylated proteins were enriched using streptavidin beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All samples were then combined and analyzed by LC-MS/MS. (B) Filtering scheme for mass spectrometric data. +R and -R refer to rapamycin, and +B and -B refer to biotin. For each dataset, proteins were first ranked by the extent of biotinylation (ratiometric analysis referencing omit biotin controls, filter 1). Next, proteins were ranked by relative proximity to ER-mitochondria contacts versus cytosol (ratiometric analysis referencing TurboID-NES, filter 2). (C) Scatterplot showing log 2 (128C/126C) (filter 1) versus log 2 (128C/130C) (filter 2) for each protein in replicate 1 of split-TurboID treated with rapamycin and biotin. Known ERM and OMM proteins (as annotated by GOCC) are labeled blue and red, respectively; ERM and OMM dual-annotated proteins are colored purple, and all other proteins are shown in black. Cutoffs used to filter the mass spectrometric data and obtain the filtered proteome are shown by dashed lines. Zoom of boxed region shown in the upper left.
    Figure Legend Snippet: Proteomic mapping of ER-mitochondria contacts in HEK293T cells. (A) Experimental design and labeling conditions for MS-based proteomics. Cells stably expressing the indicated constructs were labeled with 50 μM biotin and 100 nM rapamycin. Split-TurboID (ERM/OMM) samples were labeled for 4 hours and full-length TurboID samples were labeled for 1 minute. Cells were then lysed, and biotinylated proteins were enriched using streptavidin beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All samples were then combined and analyzed by LC-MS/MS. (B) Filtering scheme for mass spectrometric data. +R and -R refer to rapamycin, and +B and -B refer to biotin. For each dataset, proteins were first ranked by the extent of biotinylation (ratiometric analysis referencing omit biotin controls, filter 1). Next, proteins were ranked by relative proximity to ER-mitochondria contacts versus cytosol (ratiometric analysis referencing TurboID-NES, filter 2). (C) Scatterplot showing log 2 (128C/126C) (filter 1) versus log 2 (128C/130C) (filter 2) for each protein in replicate 1 of split-TurboID treated with rapamycin and biotin. Known ERM and OMM proteins (as annotated by GOCC) are labeled blue and red, respectively; ERM and OMM dual-annotated proteins are colored purple, and all other proteins are shown in black. Cutoffs used to filter the mass spectrometric data and obtain the filtered proteome are shown by dashed lines. Zoom of boxed region shown in the upper left.

    Techniques Used: Labeling, Stable Transfection, Expressing, Construct, Liquid Chromatography with Mass Spectroscopy

    Additional characterization of low-affinity split-TurboID and colocalization of organelle-targeted constructs. Related to Figure 2 . (A) Split-TurboID reconstitutes with fast kinetics. Cells transiently transfected with split-TurboID constructs were pre-treated with 100 nM rapamycin for the indicated time before labeling with biotin. Whole cell lysates were analyzed by streptavidin blotting, and anti-V5 and anti-HA staining show expression levels. Full-length TurboID (30 minutes) and full-length BioID (30 minutes and 18 hours) were included for comparison. (B) Confocal fluorescence imaging of split-TurboID targeted to the mitochondrial matrix. Cells were labeled with 500 nM MitoTracker for 30 minutes, fixed, and stained with anti-V5 or anti-HA to detect the respective fragment. Scale bars, 20 μm. Colocalization of V5 or HA with MitoTracker is quantified on the right. Quantitation from 5 fields of view per condition are included. (C) Confocal fluorescence imaging of split-TurboID (L73/G74) targeted to the ER lumen. Cells transiently transfected with the N-terminal fragment alone were fixed and stained with anti-V5 to detect the N-terminal fragment, and with CANX, which is an ER marker. Cells transiently transfected with both fragments were fixed and stained with anti-V5 and anti-HA to detect the respective fragments simultaneously. Scale bars, 20 μm. Colocalization of either V5 and CANX or V5 and HA is quantified on the right. Quantitation from 5 fields of view per condition are included.
    Figure Legend Snippet: Additional characterization of low-affinity split-TurboID and colocalization of organelle-targeted constructs. Related to Figure 2 . (A) Split-TurboID reconstitutes with fast kinetics. Cells transiently transfected with split-TurboID constructs were pre-treated with 100 nM rapamycin for the indicated time before labeling with biotin. Whole cell lysates were analyzed by streptavidin blotting, and anti-V5 and anti-HA staining show expression levels. Full-length TurboID (30 minutes) and full-length BioID (30 minutes and 18 hours) were included for comparison. (B) Confocal fluorescence imaging of split-TurboID targeted to the mitochondrial matrix. Cells were labeled with 500 nM MitoTracker for 30 minutes, fixed, and stained with anti-V5 or anti-HA to detect the respective fragment. Scale bars, 20 μm. Colocalization of V5 or HA with MitoTracker is quantified on the right. Quantitation from 5 fields of view per condition are included. (C) Confocal fluorescence imaging of split-TurboID (L73/G74) targeted to the ER lumen. Cells transiently transfected with the N-terminal fragment alone were fixed and stained with anti-V5 to detect the N-terminal fragment, and with CANX, which is an ER marker. Cells transiently transfected with both fragments were fixed and stained with anti-V5 and anti-HA to detect the respective fragments simultaneously. Scale bars, 20 μm. Colocalization of either V5 and CANX or V5 and HA is quantified on the right. Quantitation from 5 fields of view per condition are included.

    Techniques Used: Construct, Transfection, Labeling, Staining, Expressing, Fluorescence, Imaging, Quantitation Assay, Marker

    Engineering split-TurboID. (A) Schematic of split-TurboID reconstitution using the chemically-inducible FRB-FKBP dimerization system and design of constructs used to screen pairs of split sites. Upon rapamycin treatment, two inactive fragments of TurboID reconstitute to form an active enzyme capable of generating biotin-5’-AMP for promiscuous proximity-dependent labeling. N-terminal fragments (Tb(N)) were fused to FKBP and V5. C-terminal fragments (Tb(C)) were fused to HA, HaloTag, and FRB. The HaloTag was retained for initial screening as previous studies have shown that it can improve stability ( 18 ). (B) Split sites tested. Ten split sites were tested in the first round, with the 73/74 split being the best. In the second round, four additional sites were tested. Split sites are indicated as red lines along the TurboID protein sequence. The α helices are shown in blue and the β sheets are shown in purple. (C) Results of split site screen. Split-BioID (split at E256/G257) ( 20 ) and Contact-ID (split at G78/G79) ( 21 ) are previously described versions of split-BioID. Each fragment pair was tested in HEK293T cells with 24 hours biotin incubation in the presence or absence of rapamycin. At right, cells expressing full-length (FL) TurboID were incubated with biotin for 30 minutes. FL BioID was incubated with biotin for 24 hours. Cell lysates were analyzed by streptavidin blotting as in (D), and quantification was performed by dividing the streptavidin sum intensity by the anti-V5 intensity. Values were normalized to that of full-length TurboID. (D) Streptavidin blot comparing our final split-TurboID pair to full-length TurboID and BioID, and the previously-described split-BioID and Contact-ID pairs ( 20 , 21 ). Labeling conditions were same as in (D). For each construct pair, lanes are shown with both fragments present (B), N-terminal fragment only (N), or C-terminal fragment only (C). Anti-V5 and anti-HA blotting show expression levels of N-terminal fragments (V5-tagged), C-terminal fragments (HA-tagged), and full-length enzymes (V5-tagged). Dashed lines indicate separate blots performed at the same time and developed simultaneously. Asterisks indicate ligase self-biotinylation. Full blots are shown in Supplementary Figure 1 . (E) N- and C-terminal fragments (blue and purple, respectively) of split-TurboID (73/74), indicated on a structure of E. coli biotin ligase (PDB: 2EWN), from which TurboID was evolved ( 7 ). Biotin-AMP in the active site is shown in yellow. The low-affinity split-TurboID cut site is shown in red, the high-affinity split-TurboID cut site is shown in blue, and the previous split-BioID cut site is shown in black.
    Figure Legend Snippet: Engineering split-TurboID. (A) Schematic of split-TurboID reconstitution using the chemically-inducible FRB-FKBP dimerization system and design of constructs used to screen pairs of split sites. Upon rapamycin treatment, two inactive fragments of TurboID reconstitute to form an active enzyme capable of generating biotin-5’-AMP for promiscuous proximity-dependent labeling. N-terminal fragments (Tb(N)) were fused to FKBP and V5. C-terminal fragments (Tb(C)) were fused to HA, HaloTag, and FRB. The HaloTag was retained for initial screening as previous studies have shown that it can improve stability ( 18 ). (B) Split sites tested. Ten split sites were tested in the first round, with the 73/74 split being the best. In the second round, four additional sites were tested. Split sites are indicated as red lines along the TurboID protein sequence. The α helices are shown in blue and the β sheets are shown in purple. (C) Results of split site screen. Split-BioID (split at E256/G257) ( 20 ) and Contact-ID (split at G78/G79) ( 21 ) are previously described versions of split-BioID. Each fragment pair was tested in HEK293T cells with 24 hours biotin incubation in the presence or absence of rapamycin. At right, cells expressing full-length (FL) TurboID were incubated with biotin for 30 minutes. FL BioID was incubated with biotin for 24 hours. Cell lysates were analyzed by streptavidin blotting as in (D), and quantification was performed by dividing the streptavidin sum intensity by the anti-V5 intensity. Values were normalized to that of full-length TurboID. (D) Streptavidin blot comparing our final split-TurboID pair to full-length TurboID and BioID, and the previously-described split-BioID and Contact-ID pairs ( 20 , 21 ). Labeling conditions were same as in (D). For each construct pair, lanes are shown with both fragments present (B), N-terminal fragment only (N), or C-terminal fragment only (C). Anti-V5 and anti-HA blotting show expression levels of N-terminal fragments (V5-tagged), C-terminal fragments (HA-tagged), and full-length enzymes (V5-tagged). Dashed lines indicate separate blots performed at the same time and developed simultaneously. Asterisks indicate ligase self-biotinylation. Full blots are shown in Supplementary Figure 1 . (E) N- and C-terminal fragments (blue and purple, respectively) of split-TurboID (73/74), indicated on a structure of E. coli biotin ligase (PDB: 2EWN), from which TurboID was evolved ( 7 ). Biotin-AMP in the active site is shown in yellow. The low-affinity split-TurboID cut site is shown in red, the high-affinity split-TurboID cut site is shown in blue, and the previous split-BioID cut site is shown in black.

    Techniques Used: Construct, Labeling, Sequencing, Incubation, Expressing

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002061

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

    Techniques Used: Incubation, Mutagenesis

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

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

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm782

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

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

    25) Product Images from "CircPVT1 promotes gallbladder cancer growth by sponging miR-339-3p and regulates MCL-1 expression"

    Article Title: CircPVT1 promotes gallbladder cancer growth by sponging miR-339-3p and regulates MCL-1 expression

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-021-00577-y

    CircPVT1 sponges to miR-339-3p in GBC. A Binding sequence between miR-339-3p and circPVT1. B The luciferase reporter systems showed that the miR-339-3p mimic considerably reduced the luciferase activity of the circPVT1-WT luciferase reporter vector compared with the negative control, while the miR-339-3p mimic did not pose any impact on the luciferase activity of circPVT1-MUT in NOZ cells. C The miR-339-3p expression was increased after circPVT1 knockdown in NOZ and SGC-996 cells. D The miR-339-3p expression was decreased after circPVT1 overexpression in NOZ cells. E Anti-AGO2 RIP was executed in NOZ cells and qRT-PCR was detected for the enrichment of circPVT1 and miR-339-3p. F The biotin-coupled RNA complex was pulled down by incubating the cell lysate with streptavidin-coated magnetic beads and the circPVT1 was detected by qRT-PCR. G Binding sequence between miR-339-3p and MCL-1. H The luciferase reporter systems showed that the circPVT1 considerably enhanced the luciferase activity of the MCL-1 WT luciferase reporter vector compared with the negative control, while the miR-339-3p mimic reduced the luciferase activity of the MCL-1-WT luciferase reporter, but both did not pose any impact on the luciferase activity of MCL-1 MUT in NOZ and SGC-996 cells. Data are expressed as mean ± SEM. ∗ p
    Figure Legend Snippet: CircPVT1 sponges to miR-339-3p in GBC. A Binding sequence between miR-339-3p and circPVT1. B The luciferase reporter systems showed that the miR-339-3p mimic considerably reduced the luciferase activity of the circPVT1-WT luciferase reporter vector compared with the negative control, while the miR-339-3p mimic did not pose any impact on the luciferase activity of circPVT1-MUT in NOZ cells. C The miR-339-3p expression was increased after circPVT1 knockdown in NOZ and SGC-996 cells. D The miR-339-3p expression was decreased after circPVT1 overexpression in NOZ cells. E Anti-AGO2 RIP was executed in NOZ cells and qRT-PCR was detected for the enrichment of circPVT1 and miR-339-3p. F The biotin-coupled RNA complex was pulled down by incubating the cell lysate with streptavidin-coated magnetic beads and the circPVT1 was detected by qRT-PCR. G Binding sequence between miR-339-3p and MCL-1. H The luciferase reporter systems showed that the circPVT1 considerably enhanced the luciferase activity of the MCL-1 WT luciferase reporter vector compared with the negative control, while the miR-339-3p mimic reduced the luciferase activity of the MCL-1-WT luciferase reporter, but both did not pose any impact on the luciferase activity of MCL-1 MUT in NOZ and SGC-996 cells. Data are expressed as mean ± SEM. ∗ p

    Techniques Used: Binding Assay, Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Expressing, Over Expression, Quantitative RT-PCR, Magnetic Beads

    26) Product Images from "Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome"

    Article Title: Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243905

    Detection of in vitro DNA binding of polyamides 1 and 2 via Cognate Site Identification (CSI) by SELEX-seq. ( A ) Overview of CSI by SELEX-seq workflow. A randomized 20 bp DNA library is incubated with biotinylated polyamide, DNA is enriched by streptavidin-coated magnetic beads, PCR amplified and sequenced by NGS to obtain k-mers (CSI enrichment) representing polyamide-DNA binding. Enrichment is displayed as a histogram plot and high binding sequences are represented as a position weight matrix (PWM) logo. Specificity and energy landscapes (SELs) are created to visualize the full spectrum of DNA binding across all sequence permutations of an 8-mer binding site. ( B ) PWM logos for polyamides 1 ( top ) and 2 ( bottom ). ( C ) Scatterplot comparison of in vitro DNA binding for 1 vs 2 . CSI enrichment for 8-mers is plotted for sequences containing 5’-WGWWCW-3’ ( red ) and 5’-WGGWCW-3’ ( green ). ( D ) Comprehensive SELs for 1 ( top ) and 2 ( bottom ) using 5’-WGWWCW-3’ and 5’-WGGWCW-3’ as seed motif, respectively, where W = A or T.
    Figure Legend Snippet: Detection of in vitro DNA binding of polyamides 1 and 2 via Cognate Site Identification (CSI) by SELEX-seq. ( A ) Overview of CSI by SELEX-seq workflow. A randomized 20 bp DNA library is incubated with biotinylated polyamide, DNA is enriched by streptavidin-coated magnetic beads, PCR amplified and sequenced by NGS to obtain k-mers (CSI enrichment) representing polyamide-DNA binding. Enrichment is displayed as a histogram plot and high binding sequences are represented as a position weight matrix (PWM) logo. Specificity and energy landscapes (SELs) are created to visualize the full spectrum of DNA binding across all sequence permutations of an 8-mer binding site. ( B ) PWM logos for polyamides 1 ( top ) and 2 ( bottom ). ( C ) Scatterplot comparison of in vitro DNA binding for 1 vs 2 . CSI enrichment for 8-mers is plotted for sequences containing 5’-WGWWCW-3’ ( red ) and 5’-WGGWCW-3’ ( green ). ( D ) Comprehensive SELs for 1 ( top ) and 2 ( bottom ) using 5’-WGWWCW-3’ and 5’-WGGWCW-3’ as seed motif, respectively, where W = A or T.

    Techniques Used: In Vitro, Binding Assay, Incubation, Magnetic Beads, Polymerase Chain Reaction, Amplification, Next-Generation Sequencing, Sequencing

    27) Product Images from "Directed evolution of APEX2 for electron microscopy and proteomics"

    Article Title: Directed evolution of APEX2 for electron microscopy and proteomics

    Journal: Nature methods

    doi: 10.1038/nmeth.3179

    APEX2 has improved cellular activity and sensitivity for proteomic tagging and electron microscopy. ( A ) HEK cells expressing the indicated APEX variant were labeled with biotin-phenol for 1 minute before fixation and staining with streptavidin-AlexaFluor568 (red) to visualize biotinylation sites, and anti-Flag antibody (cyan) to visualize APEX expression. Arrowheads, cells with low APEX2 expression and strong biotinylation. Asterisks, cells with high APEX expression and low biotinylation. DIC, differential interference contrast. Images representative of 25 fields of view. Scale bars, 50 μm. ( B ) Quantitation of experiment shown in (A). > 50 single cells were analyzed across > 16 fields of view for each APEX variant. Mean streptavidin intensities are plotted ± 1 s. d. ( C ) Comparison of proximity dependent biotinylation by APEX2 and APEX. Live HEK cells expressing APEX or APEX2 targeted to the endoplasmic reticulum (ER) membrane (ERM) or outer mitochondrial membrane (OMM) were labeled with biotin-phenol as in (A). After cell lysis, biotinylated proteins were enriched using streptavidin beads and blotted for the endogenous ER and mitochondrial proteins shown. ( D ) Comparison of APEX2 and APEX for EM imaging of HEK cells expressing plasma membrane-targeted constructs. Arrowheads point to plasma membrane. Images representative of > 3 fields of view. Scale bars, 500 nm. For (C) and (D), controls showed that APEX and APEX2 construct pairs were expressed at similar levels (data not shown). ( E ) Purified peroxidases were incubated with 1.4 mM or 0.5 mM guaiacol (a model aromatic substrate 24 , 25 ) to mimic EM and proteomic tagging conditions, respectively. Initial rates (V o ) were measured for a range of H 2 O 2 concentrations. Each plot is representative of 2-5 trials.
    Figure Legend Snippet: APEX2 has improved cellular activity and sensitivity for proteomic tagging and electron microscopy. ( A ) HEK cells expressing the indicated APEX variant were labeled with biotin-phenol for 1 minute before fixation and staining with streptavidin-AlexaFluor568 (red) to visualize biotinylation sites, and anti-Flag antibody (cyan) to visualize APEX expression. Arrowheads, cells with low APEX2 expression and strong biotinylation. Asterisks, cells with high APEX expression and low biotinylation. DIC, differential interference contrast. Images representative of 25 fields of view. Scale bars, 50 μm. ( B ) Quantitation of experiment shown in (A). > 50 single cells were analyzed across > 16 fields of view for each APEX variant. Mean streptavidin intensities are plotted ± 1 s. d. ( C ) Comparison of proximity dependent biotinylation by APEX2 and APEX. Live HEK cells expressing APEX or APEX2 targeted to the endoplasmic reticulum (ER) membrane (ERM) or outer mitochondrial membrane (OMM) were labeled with biotin-phenol as in (A). After cell lysis, biotinylated proteins were enriched using streptavidin beads and blotted for the endogenous ER and mitochondrial proteins shown. ( D ) Comparison of APEX2 and APEX for EM imaging of HEK cells expressing plasma membrane-targeted constructs. Arrowheads point to plasma membrane. Images representative of > 3 fields of view. Scale bars, 500 nm. For (C) and (D), controls showed that APEX and APEX2 construct pairs were expressed at similar levels (data not shown). ( E ) Purified peroxidases were incubated with 1.4 mM or 0.5 mM guaiacol (a model aromatic substrate 24 , 25 ) to mimic EM and proteomic tagging conditions, respectively. Initial rates (V o ) were measured for a range of H 2 O 2 concentrations. Each plot is representative of 2-5 trials.

    Techniques Used: Activity Assay, Electron Microscopy, Expressing, Variant Assay, Labeling, Staining, Quantitation Assay, Lysis, Imaging, Construct, Purification, Incubation

    Yeast display evolution of APEX2 for electron microscopy (EM) and proteomics applications. ( A ) Structure of wild-type soybean ascorbate peroxidase (APX) with mutations present in APEX, APEX2, and VPG APEX indicated. New mutations discovered in this study are highlighted yellow. The active site is magnified to show the heme cofactor, aromatic substrate (salicylhydroxamic acid, brown) binding site, and A134 position (yellow) that is mutated to proline in APEX2. From PDB ID 1 V0H 9 . ( B ) Scheme showing how APEX or APEX2 can be used as a reporter to generate contrast for EM. 4 ( C ) Scheme showing how APEX or APEX2 can be used for proteomic tagging in living cells. 5 , 6 Blue B, biotin. ( D ) Labeling and selection scheme used to evolve APEX2 by yeast display. Biotin-phenol was added to a dilute yeast suspension for 1 minute to allow cells displaying highly active APEX variants to promiscuously biotinylate themselves. Minimal inter-cellular labeling was observed under these conditions. Biotinylation sites (blue) were stained with fluorescent streptavidin-phycoerythrin (red), and APEX expression level was quantified via anti-myc antibody staining (yellow). Two-dimensional fluorescence activated cell sorting (FACS) was used to enrich for cells displaying the highest activity/expression ratio (i.e., streptavidin/myc ratio). ( E ) FACS plot showing the initial APEX mutant library, and the sorting gate used in the first round of selection (red polygon). Single trial, 10,000 cells shown. FACS analyses of enriched populations and individual clones shown in Supplementary Figure 2 .
    Figure Legend Snippet: Yeast display evolution of APEX2 for electron microscopy (EM) and proteomics applications. ( A ) Structure of wild-type soybean ascorbate peroxidase (APX) with mutations present in APEX, APEX2, and VPG APEX indicated. New mutations discovered in this study are highlighted yellow. The active site is magnified to show the heme cofactor, aromatic substrate (salicylhydroxamic acid, brown) binding site, and A134 position (yellow) that is mutated to proline in APEX2. From PDB ID 1 V0H 9 . ( B ) Scheme showing how APEX or APEX2 can be used as a reporter to generate contrast for EM. 4 ( C ) Scheme showing how APEX or APEX2 can be used for proteomic tagging in living cells. 5 , 6 Blue B, biotin. ( D ) Labeling and selection scheme used to evolve APEX2 by yeast display. Biotin-phenol was added to a dilute yeast suspension for 1 minute to allow cells displaying highly active APEX variants to promiscuously biotinylate themselves. Minimal inter-cellular labeling was observed under these conditions. Biotinylation sites (blue) were stained with fluorescent streptavidin-phycoerythrin (red), and APEX expression level was quantified via anti-myc antibody staining (yellow). Two-dimensional fluorescence activated cell sorting (FACS) was used to enrich for cells displaying the highest activity/expression ratio (i.e., streptavidin/myc ratio). ( E ) FACS plot showing the initial APEX mutant library, and the sorting gate used in the first round of selection (red polygon). Single trial, 10,000 cells shown. FACS analyses of enriched populations and individual clones shown in Supplementary Figure 2 .

    Techniques Used: Electron Microscopy, Binding Assay, Labeling, Selection, Staining, Expressing, Fluorescence, FACS, Activity Assay, Mutagenesis, Clone Assay

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

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.00186-06

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

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

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

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

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2010.07.005

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

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

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

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

    Journal: Gene

    doi: 10.1016/j.gene.2012.12.042

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

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

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

    Techniques Used: Labeling, Sequencing, Fluorescence

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

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

    31) Product Images from "Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II"

    Article Title: Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.2000.01087.x

    Removal of B220 + cells from spleen cells depletes B cells and enriches for T cells and macrophages. Spleen cells were analysed by flow cytometry before and after removal of cells labelled with biotinylated anti-B220 using streptavidin-coated beads. B cells, T cells and macrophages were labelled with biotinylated antibodies (anti-B220, anti-CD3ε and anti-Mac-1α, respectively) and detected with streptavidin–FITC (Materials and Methods).
    Figure Legend Snippet: Removal of B220 + cells from spleen cells depletes B cells and enriches for T cells and macrophages. Spleen cells were analysed by flow cytometry before and after removal of cells labelled with biotinylated anti-B220 using streptavidin-coated beads. B cells, T cells and macrophages were labelled with biotinylated antibodies (anti-B220, anti-CD3ε and anti-Mac-1α, respectively) and detected with streptavidin–FITC (Materials and Methods).

    Techniques Used: Flow Cytometry, Cytometry

    32) Product Images from "Ancient and Recent Adaptive Evolution of Primate Non-Homologous End Joining Genes"

    Article Title: Ancient and Recent Adaptive Evolution of Primate Non-Homologous End Joining Genes

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001169

    Interactions with other repair proteins have been conserved in Nbs1 despite its positive selection. A) Positively selected residues 9 and 185 (red balls) are mapped onto the partial Nbs1 structure (PDB 3HUE) [48] . B) SNP frequencies of Q185E are reported for the ten human populations included in the HapMap project ( http://hapmap.ncbi.nlm.nih.gov/ ). Three-letter labels are standard codes (ASW - African ancestry in Southwest USA; CEU - Utah residents with Northern and Western European ancestry; CHB- Han Chinese in Beijing, China; CHD - Chinese in Metropolitan Denver, Colorado; GIH - Gujarati Indians in Houston, Texas; JPT - Japanese in Tokyo, Japan; LWK - Luhya in Webuye, Kenya; MEX - Mexican ancestry in Los Angeles, California; MKK - Maasai in Kinyawa, Kenya; TSI - Toscans in Italy). C) Binding assays were performed between recombinant biotinylated MRN complexes containing Nbs1 E185 or Q185, and an N-terminal Flag-tagged fragment of Mdc1 containing amino acids 1 to 740, as indicated. The biotinylated MRN complexes (20nM) were incubated with 45 nM Mdc1 and then isolated with streptavidin-coated magnetic beads. Bound protein was visualized by western blotting with anti-Flag (Mdc1) and anti-Nbs1 antibodies. D) MRN complexes containing Nbs1 E185 or Q185 were tested in ATM kinase assays with linear DNA as indicated. Phosphorylation of the substrate, GST-p53 (aa 1–100), was assessed by western blotting using a phospho-specific antibody directed against p53-phospho-ser15 as previously described [57] .
    Figure Legend Snippet: Interactions with other repair proteins have been conserved in Nbs1 despite its positive selection. A) Positively selected residues 9 and 185 (red balls) are mapped onto the partial Nbs1 structure (PDB 3HUE) [48] . B) SNP frequencies of Q185E are reported for the ten human populations included in the HapMap project ( http://hapmap.ncbi.nlm.nih.gov/ ). Three-letter labels are standard codes (ASW - African ancestry in Southwest USA; CEU - Utah residents with Northern and Western European ancestry; CHB- Han Chinese in Beijing, China; CHD - Chinese in Metropolitan Denver, Colorado; GIH - Gujarati Indians in Houston, Texas; JPT - Japanese in Tokyo, Japan; LWK - Luhya in Webuye, Kenya; MEX - Mexican ancestry in Los Angeles, California; MKK - Maasai in Kinyawa, Kenya; TSI - Toscans in Italy). C) Binding assays were performed between recombinant biotinylated MRN complexes containing Nbs1 E185 or Q185, and an N-terminal Flag-tagged fragment of Mdc1 containing amino acids 1 to 740, as indicated. The biotinylated MRN complexes (20nM) were incubated with 45 nM Mdc1 and then isolated with streptavidin-coated magnetic beads. Bound protein was visualized by western blotting with anti-Flag (Mdc1) and anti-Nbs1 antibodies. D) MRN complexes containing Nbs1 E185 or Q185 were tested in ATM kinase assays with linear DNA as indicated. Phosphorylation of the substrate, GST-p53 (aa 1–100), was assessed by western blotting using a phospho-specific antibody directed against p53-phospho-ser15 as previously described [57] .

    Techniques Used: Selection, Northern Blot, Western Blot, Binding Assay, Recombinant, Incubation, Isolation, Magnetic Beads

    33) Product Images from "Tuning the Drug Efflux Activity of an ABC Transporter invivo by in vitro Selected DARPin Binders"

    Article Title: Tuning the Drug Efflux Activity of an ABC Transporter invivo by in vitro Selected DARPin Binders

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037845

    Ribosome display and ELISA set-up. ( A ) Sketch delineating one DARPin selection round using ribosome display (adopted from [31] ). The DARPin library in form of mRNA is in vitro translated and stable ribosomal complexes linking the phenotype (folded DARPins) with the genotype (translated mRNA) are generated. The ribosomal complexes are allowed to bind to immobilized bLmrCD AviC . After a washing step of variable length (depending on selection stringency), bound ribosomal complexes are destabilized and mRNA encoding for potential target-specific DARPins is liberated. The eluted mRNA is amplified by reverse transcription and PCR to double stranded DNA, which is in vitro transcribed into mRNA for another round of selection or used for binder analysis. ( B ) Schematic drawing of the ELISA set up. Protein A is coated onto the ELISA well and is decorated with an anti-myc antibody that immobilizes the DARPins via the C-terminal Myc5-tag. Upon binding of purified, biotinylated target protein (e.g. LmrCD, AcrB or MsbA in our study) to DARPin, the target protein is detected using a streptavidin-alkaline phosphatase the activity of which was detected colourimetrically at OD 405 using p-nitrophenyl phosphate as a substrate.
    Figure Legend Snippet: Ribosome display and ELISA set-up. ( A ) Sketch delineating one DARPin selection round using ribosome display (adopted from [31] ). The DARPin library in form of mRNA is in vitro translated and stable ribosomal complexes linking the phenotype (folded DARPins) with the genotype (translated mRNA) are generated. The ribosomal complexes are allowed to bind to immobilized bLmrCD AviC . After a washing step of variable length (depending on selection stringency), bound ribosomal complexes are destabilized and mRNA encoding for potential target-specific DARPins is liberated. The eluted mRNA is amplified by reverse transcription and PCR to double stranded DNA, which is in vitro transcribed into mRNA for another round of selection or used for binder analysis. ( B ) Schematic drawing of the ELISA set up. Protein A is coated onto the ELISA well and is decorated with an anti-myc antibody that immobilizes the DARPins via the C-terminal Myc5-tag. Upon binding of purified, biotinylated target protein (e.g. LmrCD, AcrB or MsbA in our study) to DARPin, the target protein is detected using a streptavidin-alkaline phosphatase the activity of which was detected colourimetrically at OD 405 using p-nitrophenyl phosphate as a substrate.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Selection, In Vitro, Generated, Amplification, Polymerase Chain Reaction, Binding Assay, Purification, Activity Assay

    34) Product Images from "Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome"

    Article Title: Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome

    Journal: Scientific Reports

    doi: 10.1038/srep28324

    Design of DNA probes and experimental flow for the enrichment of HTLV-1 proviral DNA. ( A ) Design of the probes. One hundred and forty eight probes, 120 bp in length and with a 60 bp tiling, were constructed. ( B ) Experimental flow for the applications of the targeted enrichment. Genomic DNA or chromatin samples extracted from infected cells can be analyzed to render genome-wide or provirus-focused information. The enrichment of genomic DNA samples containing the viral genome may help discover new proviral sequences with more accuracy due to the higher number of reads that are obtained during the process. The enrichment of ChIP DNA could provide information on the epigenetic profile of the provirus. In addition, not enriching the samples could give the genome-wide context of the proviral profile. ( C ) Experimental flow for the enrichment protocol. DNA libraries prepared for NGS are mixed with the virus-specific biotinylated probes to allow the hybridization. Subsequently, streptavidin-coated magnetic beads are added to allow the isolation of the proviral DNA fragments.
    Figure Legend Snippet: Design of DNA probes and experimental flow for the enrichment of HTLV-1 proviral DNA. ( A ) Design of the probes. One hundred and forty eight probes, 120 bp in length and with a 60 bp tiling, were constructed. ( B ) Experimental flow for the applications of the targeted enrichment. Genomic DNA or chromatin samples extracted from infected cells can be analyzed to render genome-wide or provirus-focused information. The enrichment of genomic DNA samples containing the viral genome may help discover new proviral sequences with more accuracy due to the higher number of reads that are obtained during the process. The enrichment of ChIP DNA could provide information on the epigenetic profile of the provirus. In addition, not enriching the samples could give the genome-wide context of the proviral profile. ( C ) Experimental flow for the enrichment protocol. DNA libraries prepared for NGS are mixed with the virus-specific biotinylated probes to allow the hybridization. Subsequently, streptavidin-coated magnetic beads are added to allow the isolation of the proviral DNA fragments.

    Techniques Used: Flow Cytometry, Construct, Infection, Genome Wide, Chromatin Immunoprecipitation, Next-Generation Sequencing, Hybridization, Magnetic Beads, Isolation

    35) Product Images from "MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.01613-18

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

    Techniques Used: Transfection, Western Blot, Labeling

    36) Product Images from "Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation"

    Article Title: Phosphorylation of the RNA Polymerase II Carboxyl-Terminal Domain by CDK9 Is Directly Responsible for Human Immunodeficiency Virus Type 1 Tat-Activated Transcriptional Elongation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.13.4622-4637.2002

    Strategy used for analyzing transcription elongation complexes. (A) Structure of HIV-LTR template. DNA templates containing the lac operator (lacO) binding site for the lac repressor protein (LacR) and a terminator (τ) sequence were biotinylated and bound to streptavidin beads. (B) Elongation complexes were trapped by the lac repressor (LacR) after incubation of the immobilized templates with HeLa nuclear extract (NE) in the presence of nucleotide triphosphates and LacR and in the absence or presence of Tat. The CTD of the RNA polymerase was phosphorylated during the elongation reaction due to the activity of CDK7 and CDK9. (C) Elongation complexes arrested by LacR were treated with PP1 to remove phosphate groups from the CTD. (D) The phosphatase-treated complexes can resume transcription elongation after the addition of nucleotides and IPTG. During the chase reaction the CTD became phosphorylated by CDK9. The addition of DRB blocked the rephosphorylation of the CTD and induced pausing of the transcription complex at the terminator sequences.
    Figure Legend Snippet: Strategy used for analyzing transcription elongation complexes. (A) Structure of HIV-LTR template. DNA templates containing the lac operator (lacO) binding site for the lac repressor protein (LacR) and a terminator (τ) sequence were biotinylated and bound to streptavidin beads. (B) Elongation complexes were trapped by the lac repressor (LacR) after incubation of the immobilized templates with HeLa nuclear extract (NE) in the presence of nucleotide triphosphates and LacR and in the absence or presence of Tat. The CTD of the RNA polymerase was phosphorylated during the elongation reaction due to the activity of CDK7 and CDK9. (C) Elongation complexes arrested by LacR were treated with PP1 to remove phosphate groups from the CTD. (D) The phosphatase-treated complexes can resume transcription elongation after the addition of nucleotides and IPTG. During the chase reaction the CTD became phosphorylated by CDK9. The addition of DRB blocked the rephosphorylation of the CTD and induced pausing of the transcription complex at the terminator sequences.

    Techniques Used: Binding Assay, Sequencing, Incubation, Activity Assay

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

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

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2016.0167

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

    Techniques Used: Magnetic Beads, Cell Culture, Mutagenesis

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm782

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

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

    39) Product Images from "Cholera toxin inhibits IL-12 production and CD8?+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function"

    Article Title: Cholera toxin inhibits IL-12 production and CD8?+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20080912

    CT and dbcAMP reduce IRF8 binding to the ISRE-like region of the mouse IL-12p40 promoter and prevent IRF1–IRF8 heterocomplex formation. (A) Schematic representation of the −92 to +12 region of the mouse IL-12p40 promoter. The ISRE-like sequence is represented in bold. (B) CT and dbcAMP, but not CTB, inhibited IRF8, but not IRF1 or TFIIB, interaction with the mouse IL-12p40 promoter. A biotinylated DNA probe corresponding to the −81 to −24 region of the mouse IL-12p40 promoter encompassing the ISRE-like sequence, and a biotinylated mutant probe containing substitution (indicated by the lower case letters) in the context of the ISRE-like element were conjugated with streptavidin-bound magnetic beads and incubated with 500 µg of nuclear extracts from RAW 264.7 cells stimulated for 4 h as indicated. CT, CTB (both at 20 ng/ml), and dbcAMP (100 µM) were added 1 h before addition of LPS (1 µg/ml) and IFN-γ (100 ng/ml). Bound material was eluted, separated by 10% SDS-PAGE, and detected by Western blot analysis using rabbit anti-IRF8 antibody. (C) CT or dbcAMP did not modify the total amount of IRF8 or IRF1 in the nucleus. 500 µg of nuclear extracts were analyzed to measure the presence of IRF8 or IRF1 by SDS-PAGE Western blot. Membranes were stripped and reblotted to verify equal protein loading using an antinucleolin antibody. (D) CT reduces IRF1–IRF8 heterocomplex formation induced by LPS and IFN-γ. 1 mg of nuclear extract was immunoprecipitated with anti-IRF1 antibody and analyzed by Western blot for the presence of IRF8 or IRF1. (E) Tyrosine phosphorylation of IRF1 is not affected by CT. 1 mg of nuclear extracts was immunoprecipitated with antiphosphotyrosine antibody and analyzed by Western blot for the presence of IRF1. Data shown are representative of at least three experiments.
    Figure Legend Snippet: CT and dbcAMP reduce IRF8 binding to the ISRE-like region of the mouse IL-12p40 promoter and prevent IRF1–IRF8 heterocomplex formation. (A) Schematic representation of the −92 to +12 region of the mouse IL-12p40 promoter. The ISRE-like sequence is represented in bold. (B) CT and dbcAMP, but not CTB, inhibited IRF8, but not IRF1 or TFIIB, interaction with the mouse IL-12p40 promoter. A biotinylated DNA probe corresponding to the −81 to −24 region of the mouse IL-12p40 promoter encompassing the ISRE-like sequence, and a biotinylated mutant probe containing substitution (indicated by the lower case letters) in the context of the ISRE-like element were conjugated with streptavidin-bound magnetic beads and incubated with 500 µg of nuclear extracts from RAW 264.7 cells stimulated for 4 h as indicated. CT, CTB (both at 20 ng/ml), and dbcAMP (100 µM) were added 1 h before addition of LPS (1 µg/ml) and IFN-γ (100 ng/ml). Bound material was eluted, separated by 10% SDS-PAGE, and detected by Western blot analysis using rabbit anti-IRF8 antibody. (C) CT or dbcAMP did not modify the total amount of IRF8 or IRF1 in the nucleus. 500 µg of nuclear extracts were analyzed to measure the presence of IRF8 or IRF1 by SDS-PAGE Western blot. Membranes were stripped and reblotted to verify equal protein loading using an antinucleolin antibody. (D) CT reduces IRF1–IRF8 heterocomplex formation induced by LPS and IFN-γ. 1 mg of nuclear extract was immunoprecipitated with anti-IRF1 antibody and analyzed by Western blot for the presence of IRF8 or IRF1. (E) Tyrosine phosphorylation of IRF1 is not affected by CT. 1 mg of nuclear extracts was immunoprecipitated with antiphosphotyrosine antibody and analyzed by Western blot for the presence of IRF1. Data shown are representative of at least three experiments.

    Techniques Used: Binding Assay, Sequencing, CtB Assay, Mutagenesis, Magnetic Beads, Incubation, SDS Page, Western Blot, Immunoprecipitation

    40) Product Images from "SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription"

    Article Title: SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx053

    The N-terminal region of SIRT7 mediates interactions with RNA and proteins. ( A ) The N-terminal part of SIRT7 is required for RNA binding. Nuclear lysates from HEK293T cells expressing Flag-tagged SIRT7 (WT), SIRT7/ΔN32 or SIRT7/ΔN78 were incubated with streptavidin-coated Dynabeads (-RNA) or with Dynabeads containing 5΄ETS-RNA (+RNA). Bound SIRT7 was analyzed on immunoblots (upper panels). Alternatively, bead-bound RNA was incubated with GST-tagged SIRT7/1-81, and binding was monitored with anti-GST antibodies (bottom panel). A scheme illustrating the domain structure of SIRT7 and the deletion mutants is shown above. See also Supplementary Figures S2A and S2B . ( B ) Pull-down assay showing impaired binding of SIRT7/ΔN78 to RNA. Bead-bound Flag-SIRT7 or the indicated deletion mutants were incubated with radiolabeled 5΄ETS-RNA (+10/+389) and SIRT7-associated RNA was analyzed by gel electrophoresis and PhosphorImaging. RNA bound to beads-only served as a negative control (beads). See also Supplementary Figure S2C . ( C ) RNA-immunoprecipitation (CLIP) showing that the N-terminal region of SIRT7 mediates RNA binding in vivo . UV-crosslinked Flag-SIRT7-RNA complexes were captured on anti-Flag beads, and co-precipitated RNA was analyzed by RT-qPCR. The percentage of precipitated RNA relative to input RNA is shown. Error bars denote means ±SD ( n = 3) (* P
    Figure Legend Snippet: The N-terminal region of SIRT7 mediates interactions with RNA and proteins. ( A ) The N-terminal part of SIRT7 is required for RNA binding. Nuclear lysates from HEK293T cells expressing Flag-tagged SIRT7 (WT), SIRT7/ΔN32 or SIRT7/ΔN78 were incubated with streptavidin-coated Dynabeads (-RNA) or with Dynabeads containing 5΄ETS-RNA (+RNA). Bound SIRT7 was analyzed on immunoblots (upper panels). Alternatively, bead-bound RNA was incubated with GST-tagged SIRT7/1-81, and binding was monitored with anti-GST antibodies (bottom panel). A scheme illustrating the domain structure of SIRT7 and the deletion mutants is shown above. See also Supplementary Figures S2A and S2B . ( B ) Pull-down assay showing impaired binding of SIRT7/ΔN78 to RNA. Bead-bound Flag-SIRT7 or the indicated deletion mutants were incubated with radiolabeled 5΄ETS-RNA (+10/+389) and SIRT7-associated RNA was analyzed by gel electrophoresis and PhosphorImaging. RNA bound to beads-only served as a negative control (beads). See also Supplementary Figure S2C . ( C ) RNA-immunoprecipitation (CLIP) showing that the N-terminal region of SIRT7 mediates RNA binding in vivo . UV-crosslinked Flag-SIRT7-RNA complexes were captured on anti-Flag beads, and co-precipitated RNA was analyzed by RT-qPCR. The percentage of precipitated RNA relative to input RNA is shown. Error bars denote means ±SD ( n = 3) (* P

    Techniques Used: RNA Binding Assay, Expressing, Incubation, Western Blot, Binding Assay, Pull Down Assay, Nucleic Acid Electrophoresis, Negative Control, Immunoprecipitation, Cross-linking Immunoprecipitation, In Vivo, Quantitative RT-PCR

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    Incubation:

    Article Title: LINC01089 suppresses lung adenocarcinoma cell proliferation and migration via miR-301b-3p/STARD13 axis
    Article Snippet: .. Biotinylated LINC01089 probes (LINC01089 biotin probe) were incubated with cell extracts and streptavidin magnetic beads (Invitrogen). ..

    Article Title: LncRNA NORAD Promotes Vascular Endothelial Cell Injury and Atherosclerosis Through Suppressing VEGF Gene Transcription via Enhancing H3K9 Deacetylation by Recruiting HDAC6
    Article Snippet: .. Following the incubation with probes, streptavidin magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleaned five times by washing buffer at 35°C. ..


    Article Title: Inactivation of the tumor suppressor p53 by long noncoding RNA RMRP
    Article Snippet: .. Protein extracts of 1 mg were
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    Article Title: Internalisation and Biological Activity of Nucleic Acids Delivering Cell-Penetrating Peptide Nanoparticles Is Controlled by the Biomolecular Corona
    Article Snippet: .. PierceTM Streptavidin superparamagnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) were washed with 0.1% Tween-20 in TBS before incubation with 1 µg of biotinylated pGL3 or 200 nM biotinylated SCO in MQ water in 1/10th of the final volume for 15 min at 37 °C with mild shaking (300 rpm). ..

    Article Title: MYC-targeted WDR4 promotes proliferation, metastasis, and sorafenib resistance by inducing CCNB1 translation in hepatocellular carcinoma
    Article Snippet: .. The biotinylated RNA was incubated with streptavidin magnetic beads (Invitrogen) overnight at 4 °C according to the manufacturer’s instructions (Thermo Scientific). ..

    Magnetic Beads:

    Article Title: LINC01089 suppresses lung adenocarcinoma cell proliferation and migration via miR-301b-3p/STARD13 axis
    Article Snippet: .. Biotinylated LINC01089 probes (LINC01089 biotin probe) were incubated with cell extracts and streptavidin magnetic beads (Invitrogen). ..

    Article Title: LncRNA NORAD Promotes Vascular Endothelial Cell Injury and Atherosclerosis Through Suppressing VEGF Gene Transcription via Enhancing H3K9 Deacetylation by Recruiting HDAC6
    Article Snippet: .. Following the incubation with probes, streptavidin magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleaned five times by washing buffer at 35°C. ..


    Article Title: Inactivation of the tumor suppressor p53 by long noncoding RNA RMRP
    Article Snippet: .. Protein extracts of 1 mg were
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    Article Title: Cholesterol is required for transcriptional repression by BASP1
    Article Snippet: .. The nuclei were then washed in buffer A and immediately used for preparation of nuclear extract for immunoprecipitation (using streptavidin magnetic beads, Thermo Fisher, 88816), chromatin immunoprecipitation (Click-ChIP with streptavidin magnetic beads), or immunofluorescence (using Streptavidin Alexa Fluor 594 conjugate, Thermo Fisher, S32356). ..

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    Binding Assay:


    other:

    Article Title: A clinically validated human saliva metatranscriptomic test for global systems biology studies
    Article Snippet: Briefly, biotinylated DNA probes with sequences complementary to human and microbial rRNAs were added to total RNA, the mixture was heated and cooled, and the probe-rRNA complexes were removed using magnetic streptavidin beads.

    Immunoprecipitation:

    Article Title: Cholesterol is required for transcriptional repression by BASP1
    Article Snippet: .. The nuclei were then washed in buffer A and immediately used for preparation of nuclear extract for immunoprecipitation (using streptavidin magnetic beads, Thermo Fisher, 88816), chromatin immunoprecipitation (Click-ChIP with streptavidin magnetic beads), or immunofluorescence (using Streptavidin Alexa Fluor 594 conjugate, Thermo Fisher, S32356). ..

    Chromatin Immunoprecipitation:

    Article Title: Cholesterol is required for transcriptional repression by BASP1
    Article Snippet: .. The nuclei were then washed in buffer A and immediately used for preparation of nuclear extract for immunoprecipitation (using streptavidin
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    Immunofluorescence:

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    Thermo Fisher streptavidin coated magnetic beads
    Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent <t>Streptavidin)</t> targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.
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    Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent Streptavidin) targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.

    Journal: Lab on a chip

    Article Title: Integration of sample preparation and analysis on an optofluidic chip for multi-target disease detection

    doi: 10.1039/c8lc00966j

    Figure Lengend Snippet: Target differentiation through dual protein-nucleic acid assay: Bar plots indicate relative number of peaks observed in the protein (green) and nucleic acid (red) channel in each of the 4 separate tests. Each bar plot is normalized with respect to the number of peaks observed with Zika nucleic acid and protein targets. Very few peaks are seen when the wrong nucleic acid (Ebola) target and wrong protein (Monovalent Streptavidin) targets are introduced. However, a significant signal is observed with dengue protein targets due to cross reactivity with Zika NS1 antibodies, but the low signal in the nucleic acid channel confirms the absence of the Zika target.

    Article Snippet: Bio One set of antibodies was labelled with the Cyanine 3 (Cy-3) fluorophore (Lumiprobe) to act as the protein specific fluorescent probe, while another set of the antibodies was modified with biotin for subsequent conjugation in 1x PBS to the streptavidin coated magnetic beads (Dynabeads™ MyOne™ Streptavidin T1, Thermofisher Scientific).

    Techniques: Acid Assay

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

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2018.02.012

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

    Article Snippet: The biotin-coupled RNA complex was pulled down by incubating the cell lysates with streptavidin-coated magnetic beads (Life Technologies).

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

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

    Journal: Gene Expression

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

    doi:

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

    Article Snippet: Streptavidin-coated magnetic beads (20 μ g) (Dynabeads, Dynal) were added to the incubation reaction and incubated for 5 min at room temperature.

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

    RNA degradation rate between PD-MK and Std conditions did not show significant difference. (A) Schematic representation of the experiment for examining RNA degradation rate. WT mESCs were transiently treated with 4-thiouridine (4sU). By this transient treatment, 4sUs were incorporated into newly synthesized RNA. RNA was then extracted from the cells, and a known amount of spike-in RNA that does not contain 4sU was added. Thereafter, the RNA mix was biotinylated. Then, from a part of this biotinylated RNA mix, biotinylated RNA was removed using streptavidin beads, and unbiotinylated RNAs that were transcribed before the addition of 4sU were recovered. RNA samples that were not treated with streptavidin beads contained both existing RNA and newly synthesized RNA. Therefore, we refer to these RNA samples as total RNAs. These were reverse transcribed and analyzed by qPCR. (B) A bar graph of the ratio of unbound and total RNA. For many samples, the ratio has exceeded 1, which was theoretically impossible. It is considered that the reverse transcription efficiency of 4sU-introduced RNA could be extremely low (see Material and Methods). (C) We assumed that the presence of biotinylated RNA during reverse transcription may trap reverse transcriptase, and that the efficiency of reverse transcription is further reduced globally. We assume that the global suppression effect of reverse transcriptase trapping is I g (global inhibitory effect). Moreover, the reverse transcription inhibitory effect of biotinylated RNA itself was defined as I s (see Material and Methods). In order to determine the appropriate value of I s , several values were assigned to I s , and mRNA half-lives in the Std condition were compared with the previously reported mRNA half-lives ( 21 ). We found that the scaling of mRNA half-lives in the Std condition and that of previously reported mRNA half-lives were getting closer when I s was 0.1. (D) Average mRNA half-life. The half-life of mRNA was calculated using the ratio obtained in (B) and the correction formula (see Material and Methods). In all genes analyzed, there was no significant difference between PD-MK and Std conditions.

    Journal: bioRxiv

    Article Title: Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

    doi: 10.1101/736207

    Figure Lengend Snippet: RNA degradation rate between PD-MK and Std conditions did not show significant difference. (A) Schematic representation of the experiment for examining RNA degradation rate. WT mESCs were transiently treated with 4-thiouridine (4sU). By this transient treatment, 4sUs were incorporated into newly synthesized RNA. RNA was then extracted from the cells, and a known amount of spike-in RNA that does not contain 4sU was added. Thereafter, the RNA mix was biotinylated. Then, from a part of this biotinylated RNA mix, biotinylated RNA was removed using streptavidin beads, and unbiotinylated RNAs that were transcribed before the addition of 4sU were recovered. RNA samples that were not treated with streptavidin beads contained both existing RNA and newly synthesized RNA. Therefore, we refer to these RNA samples as total RNAs. These were reverse transcribed and analyzed by qPCR. (B) A bar graph of the ratio of unbound and total RNA. For many samples, the ratio has exceeded 1, which was theoretically impossible. It is considered that the reverse transcription efficiency of 4sU-introduced RNA could be extremely low (see Material and Methods). (C) We assumed that the presence of biotinylated RNA during reverse transcription may trap reverse transcriptase, and that the efficiency of reverse transcription is further reduced globally. We assume that the global suppression effect of reverse transcriptase trapping is I g (global inhibitory effect). Moreover, the reverse transcription inhibitory effect of biotinylated RNA itself was defined as I s (see Material and Methods). In order to determine the appropriate value of I s , several values were assigned to I s , and mRNA half-lives in the Std condition were compared with the previously reported mRNA half-lives ( 21 ). We found that the scaling of mRNA half-lives in the Std condition and that of previously reported mRNA half-lives were getting closer when I s was 0.1. (D) Average mRNA half-life. The half-life of mRNA was calculated using the ratio obtained in (B) and the correction formula (see Material and Methods). In all genes analyzed, there was no significant difference between PD-MK and Std conditions.

    Article Snippet: For removing of biotinylated 4sU-RNA, streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin C1 beads, ThermoFisher) were used according to the manufacturer’s manual.

    Techniques: Synthesized, Real-time Polymerase Chain Reaction