fluorophore  (New England Biolabs)


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    Name:
    SNAP Surface 549
    Description:
    SNAP Surface 549 50 nmol
    Catalog Number:
    s9112s
    Price:
    344
    Category:
    Fluorochromes
    Size:
    50 nmol
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    Structured Review

    New England Biolabs fluorophore
    SNAP Surface 549
    SNAP Surface 549 50 nmol
    https://www.bioz.com/result/fluorophore/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fluorophore - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "Evolution and Characterization of a Benzylguanine-binding RNA Aptamer"

    Article Title: Evolution and Characterization of a Benzylguanine-binding RNA Aptamer

    Journal: Chemical communications (Cambridge, England)

    doi: 10.1039/c5cc07605f

    Cartoon of a bG-binding RNA aptamer and results from SELEX. (A) A bG-binding RNA aptamer can be used to target small molecule derivatives of bG to RNAs of interest. R represents a functional group such as a fluorophore or biotin. (B) Quantification of the fraction of RNA retained on bG agarose following each round of SELEX.
    Figure Legend Snippet: Cartoon of a bG-binding RNA aptamer and results from SELEX. (A) A bG-binding RNA aptamer can be used to target small molecule derivatives of bG to RNAs of interest. R represents a functional group such as a fluorophore or biotin. (B) Quantification of the fraction of RNA retained on bG agarose following each round of SELEX.

    Techniques Used: Binding Assay, Functional Assay

    FP competition assay for monitoring specificity of JX1 for bG. (A) Cartoon showing how an effective competitor of Atto488-bG for JX1 binding prevents binding of the RNA to the fluorophore results in low FP. (B) Results from the competition assay for selected guanine-containing molecules. High ΔFP values result from Atto488-bG binding to JX1 and lack of inhibition from the competitor. Each bar graph represents the average from three separate experiments and error bars represent ± S.D. N.D., Not Determined.
    Figure Legend Snippet: FP competition assay for monitoring specificity of JX1 for bG. (A) Cartoon showing how an effective competitor of Atto488-bG for JX1 binding prevents binding of the RNA to the fluorophore results in low FP. (B) Results from the competition assay for selected guanine-containing molecules. High ΔFP values result from Atto488-bG binding to JX1 and lack of inhibition from the competitor. Each bar graph represents the average from three separate experiments and error bars represent ± S.D. N.D., Not Determined.

    Techniques Used: Competitive Binding Assay, Binding Assay, Inhibition

    2) Product Images from "Dynamics and consequences of spliceosome E complex formation"

    Article Title: Dynamics and consequences of spliceosome E complex formation

    Journal: eLife

    doi: 10.7554/eLife.27592

    Counting statistics for the number of steps observed during loss of fluorescence from either U1-double DHFR or BBP-SNAP f binding events in three-color CoSMoS experiments. ( A ) Number of steps observed during loss of U1 fluorescence. Each U1 molecule contains two fluorophores, which can result in either 1 or two steps. 91% of U1 binding events are consistent with presence of only a single U1 molecule on the pre-mRNA. ( B ) Number of steps observed during loss of BBP fluorescence. Each BBP molecule contains a single fluorophore. 88% of BBP binding events are consistent with presence of only a single BBP molecule on the pre-mRNA.
    Figure Legend Snippet: Counting statistics for the number of steps observed during loss of fluorescence from either U1-double DHFR or BBP-SNAP f binding events in three-color CoSMoS experiments. ( A ) Number of steps observed during loss of U1 fluorescence. Each U1 molecule contains two fluorophores, which can result in either 1 or two steps. 91% of U1 binding events are consistent with presence of only a single U1 molecule on the pre-mRNA. ( B ) Number of steps observed during loss of BBP fluorescence. Each BBP molecule contains a single fluorophore. 88% of BBP binding events are consistent with presence of only a single BBP molecule on the pre-mRNA.

    Techniques Used: Fluorescence, Binding Assay

    Single molecule analysis of BBP-SNAP f binding dynamics. ( A ) Cartoon of a two-color CoSMoS experiment for observing BBP binding dynamics. BBP was labeled with a single green-excited fluorophore while RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Graphic representation of capped RNAs with variable BS and ΨBS used in these experiments and their corresponding label number. RNA sequences are given in Supplementary file 1 . ( C ) Bar graph comparing the relative number of BBP binding events observed on each RNA depicted in panel ( B ). ( D ) Rastergram depicting BBP binding events on RNAs containing both a BS and ΨBS (RNA 3). ( E ) Rastergram depicting BBP binding events on RNAs containing only the BS (RNA 9). ( F ) Rastergram depicting BBP binding events on RNAs containing only the ΨBS (RNA 10). ( G ) Probability density histogram of dwell times for BBP on RNAs with variable BS and ΨBS. Lines represent fits of the distributions of dwell times to equations containing one or two exponential terms. ( H–J ) Bar graph comparison of the fit parameters (τ 1 , panel H; τ 2 , panel I; the τ 2 amplitude A 2 , panel J) obtained from analysis of the dwell time distributions of BBP binding events on RNAs 3, 9, and 10. Details of the fit parameters for data shown in ( H–J ) can be found in Supplementary file 3 . Error bars in ( C, G ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( H–J ) represent the fit parameters ± S.D.
    Figure Legend Snippet: Single molecule analysis of BBP-SNAP f binding dynamics. ( A ) Cartoon of a two-color CoSMoS experiment for observing BBP binding dynamics. BBP was labeled with a single green-excited fluorophore while RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Graphic representation of capped RNAs with variable BS and ΨBS used in these experiments and their corresponding label number. RNA sequences are given in Supplementary file 1 . ( C ) Bar graph comparing the relative number of BBP binding events observed on each RNA depicted in panel ( B ). ( D ) Rastergram depicting BBP binding events on RNAs containing both a BS and ΨBS (RNA 3). ( E ) Rastergram depicting BBP binding events on RNAs containing only the BS (RNA 9). ( F ) Rastergram depicting BBP binding events on RNAs containing only the ΨBS (RNA 10). ( G ) Probability density histogram of dwell times for BBP on RNAs with variable BS and ΨBS. Lines represent fits of the distributions of dwell times to equations containing one or two exponential terms. ( H–J ) Bar graph comparison of the fit parameters (τ 1 , panel H; τ 2 , panel I; the τ 2 amplitude A 2 , panel J) obtained from analysis of the dwell time distributions of BBP binding events on RNAs 3, 9, and 10. Details of the fit parameters for data shown in ( H–J ) can be found in Supplementary file 3 . Error bars in ( C, G ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( H–J ) represent the fit parameters ± S.D.

    Techniques Used: Binding Assay, Labeling

    Single molecule analysis of U1 binding after ablation of the 5' end of the snRNA. ( A ) Cartoon of a two-color CoSMoS experiment for observing U1 binding dynamics. U1 was labeled with two green-excited fluorophores while the RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Confirmation of U1 ablation by primer extension. Primer extension of the U2 snRNA is included as a loading control. Quantification of the band intensities indicate that ≥96% of the U1 snRNA was cleaved by RNase H. ( C ) Bar graph comparing the relative number of U1 binding events observed on RNAs 3 or 7, in the presence or absence of CA and/or the RNase H ablation oligo. ( D–F ) Bar graph comparison of the fit parameters (τ 1 , panel D; τ 2 , panel E; the τ 2 amplitude A 2 , panel F) obtained from analysis of the dwell time distributions of U1 binding events. Details of the fit parameters for data shown in ( D–F ) can be found in Supplementary file 2 . Error bars in ( C ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( D–F ) represent the fit parameters ± S.D.
    Figure Legend Snippet: Single molecule analysis of U1 binding after ablation of the 5' end of the snRNA. ( A ) Cartoon of a two-color CoSMoS experiment for observing U1 binding dynamics. U1 was labeled with two green-excited fluorophores while the RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Confirmation of U1 ablation by primer extension. Primer extension of the U2 snRNA is included as a loading control. Quantification of the band intensities indicate that ≥96% of the U1 snRNA was cleaved by RNase H. ( C ) Bar graph comparing the relative number of U1 binding events observed on RNAs 3 or 7, in the presence or absence of CA and/or the RNase H ablation oligo. ( D–F ) Bar graph comparison of the fit parameters (τ 1 , panel D; τ 2 , panel E; the τ 2 amplitude A 2 , panel F) obtained from analysis of the dwell time distributions of U1 binding events. Details of the fit parameters for data shown in ( D–F ) can be found in Supplementary file 2 . Error bars in ( C ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( D–F ) represent the fit parameters ± S.D.

    Techniques Used: Binding Assay, Labeling

    Related Articles

    Labeling:

    Article Title: Dynamics and consequences of spliceosome E complex formation
    Article Snippet: Preparation and labeling of yeast whole-cell splicing extracts Yeast whole cell extract (WCE) was prepared as previously described ( ). .. SNAPf -tagged proteins were labeled by incubation of the lysate for 30 min at room temperature with the fluorophore (e.g., benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs) before gel filtration. .. A fluorophore concentration of 1.1 µM was used to label SNAPf tags.

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
    Article Snippet: Oligonucleotides (IDT Ultramers) for PCR had 50 nt of homology to the target gene C-terminal coding region and 20 nt of homology to the fusion cassette. .. In-frame fusion protein expression and stability was confirmed by immunoblotting for the target protein (e.g., ), and in the case of SNAP fusions, by SDS-PAGE after labeling with SNAP-Surface 549 (New England Biolabs). ..

    Article Title: Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging
    Article Snippet: Arp2/3-SNAP was purified from an S. cerevisiae strain in which the ARC18 locus was modified to produce an Arc18-SNAP fusion protein. .. The complex was labeled in vitro with the orange-emitting fluorescent dye DY549 (SNAP-Surface 549; New England BioLabs). .. The resulting Arp2/3-SNAP549 retained full activity of wild-type Arp2/3 complex to stimulate actin assembly in vitro ( ).

    Article Title: Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
    Article Snippet: The lysate buffer was exchanged into 50 mM HEPES/KOH pH 7.9, 50 mM KCl, 10% (v/v) glycerol, and 1 mM DTT by gel filtration using a previously published protocol , aliquoted (42 µL), frozen in liquid N2 , and stored at -80°C. .. In cases where SNAP-tagged proteins were labeled, the lysate was incubated for 30 min at room temperature with the fluorophore (e.g. , benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs; Ipswich, MA) prior to gel filtration. .. A fluorophore concentration of 2.1 µM was used to label SNAP tags, and fast SNAP (SNAPf ) tags were labeled using 1.1 µM fluorophore.

    Incubation:

    Article Title: Dynamics and consequences of spliceosome E complex formation
    Article Snippet: Preparation and labeling of yeast whole-cell splicing extracts Yeast whole cell extract (WCE) was prepared as previously described ( ). .. SNAPf -tagged proteins were labeled by incubation of the lysate for 30 min at room temperature with the fluorophore (e.g., benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs) before gel filtration. .. A fluorophore concentration of 1.1 µM was used to label SNAPf tags.

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
    Article Snippet: For SNAP fusion strains, the protocol was modified in that nuclear protein pellets were resuspended in 1–2 mL of buffer C′ (20 mM HEPES, pH 7.6, 10 mM MgSO4 , 1 mM EGTA, 10% glycerol, 3 mM DTT, and 1 μg ml−1 each of aprotinin, leupeptin, pepstatin A, and antipain). (In pilot experiments, the 20% glycerol concentration and PMSF used in ( ) were found to reduce SNAP labeling.) .. To fluorescently label extracts with SNAP fusion proteins, SNAP-Surface 549 was added with gentle vortexing to the resuspended pellet at a final concentration of 0.5 μM (unless otherwise specified), and the mixture was incubated at 4°C for 1 hr on a rotator. ..

    Article Title: Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
    Article Snippet: The lysate buffer was exchanged into 50 mM HEPES/KOH pH 7.9, 50 mM KCl, 10% (v/v) glycerol, and 1 mM DTT by gel filtration using a previously published protocol , aliquoted (42 µL), frozen in liquid N2 , and stored at -80°C. .. In cases where SNAP-tagged proteins were labeled, the lysate was incubated for 30 min at room temperature with the fluorophore (e.g. , benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs; Ipswich, MA) prior to gel filtration. .. A fluorophore concentration of 2.1 µM was used to label SNAP tags, and fast SNAP (SNAPf ) tags were labeled using 1.1 µM fluorophore.

    Filtration:

    Article Title: Dynamics and consequences of spliceosome E complex formation
    Article Snippet: Preparation and labeling of yeast whole-cell splicing extracts Yeast whole cell extract (WCE) was prepared as previously described ( ). .. SNAPf -tagged proteins were labeled by incubation of the lysate for 30 min at room temperature with the fluorophore (e.g., benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs) before gel filtration. .. A fluorophore concentration of 1.1 µM was used to label SNAPf tags.

    Article Title: Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
    Article Snippet: The lysate buffer was exchanged into 50 mM HEPES/KOH pH 7.9, 50 mM KCl, 10% (v/v) glycerol, and 1 mM DTT by gel filtration using a previously published protocol , aliquoted (42 µL), frozen in liquid N2 , and stored at -80°C. .. In cases where SNAP-tagged proteins were labeled, the lysate was incubated for 30 min at room temperature with the fluorophore (e.g. , benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs; Ipswich, MA) prior to gel filtration. .. A fluorophore concentration of 2.1 µM was used to label SNAP tags, and fast SNAP (SNAPf ) tags were labeled using 1.1 µM fluorophore.

    Concentration Assay:

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
    Article Snippet: For SNAP fusion strains, the protocol was modified in that nuclear protein pellets were resuspended in 1–2 mL of buffer C′ (20 mM HEPES, pH 7.6, 10 mM MgSO4 , 1 mM EGTA, 10% glycerol, 3 mM DTT, and 1 μg ml−1 each of aprotinin, leupeptin, pepstatin A, and antipain). (In pilot experiments, the 20% glycerol concentration and PMSF used in ( ) were found to reduce SNAP labeling.) .. To fluorescently label extracts with SNAP fusion proteins, SNAP-Surface 549 was added with gentle vortexing to the resuspended pellet at a final concentration of 0.5 μM (unless otherwise specified), and the mixture was incubated at 4°C for 1 hr on a rotator. ..

    Expressing:

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
    Article Snippet: Oligonucleotides (IDT Ultramers) for PCR had 50 nt of homology to the target gene C-terminal coding region and 20 nt of homology to the fusion cassette. .. In-frame fusion protein expression and stability was confirmed by immunoblotting for the target protein (e.g., ), and in the case of SNAP fusions, by SDS-PAGE after labeling with SNAP-Surface 549 (New England Biolabs). ..

    SDS Page:

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
    Article Snippet: Oligonucleotides (IDT Ultramers) for PCR had 50 nt of homology to the target gene C-terminal coding region and 20 nt of homology to the fusion cassette. .. In-frame fusion protein expression and stability was confirmed by immunoblotting for the target protein (e.g., ), and in the case of SNAP fusions, by SDS-PAGE after labeling with SNAP-Surface 549 (New England Biolabs). ..

    In Vitro:

    Article Title: Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging
    Article Snippet: Arp2/3-SNAP was purified from an S. cerevisiae strain in which the ARC18 locus was modified to produce an Arc18-SNAP fusion protein. .. The complex was labeled in vitro with the orange-emitting fluorescent dye DY549 (SNAP-Surface 549; New England BioLabs). .. The resulting Arp2/3-SNAP549 retained full activity of wild-type Arp2/3 complex to stimulate actin assembly in vitro ( ).

    Article Title: A ligand-based system for receptor-specific delivery of proteins
    Article Snippet: .. In-vitro labelling For keratinocyte labelling, IL-31K138A SNAP, NGFR121W SNAP or SNAP were coupled with an excess of BG-Surface549 at a 1:1.5 molar ratio (NEB # S9112) for 2 hours at 37 °C in PBS (pH 7.4) or CIB buffer (NaCl 140 mM; KCl 4 mM; CaCl2 2 mM; MgCl2 1 mM; NaOH 4.55 mM; Glucose 5 mM; HEPES 10 mM; pH 7.4). .. The coupling reaction was passed through a PD MiniTrap G-25 column (GE Healthcare #28-9180-07) to remove the excess of unbound BG.

    Microscopy:

    Article Title: Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists
    Article Snippet: Briefly, HEK293-SNAP-GLP-1R cells were transfected with PKA activation FRET biosensor AKAR4-NES (a gift from Dr. Jin Zhang, Addgene plasmid #61620) for 36 h, and FRET signal was recorded before and after ligand addition and expressed ratiometrically after normalisation to well baseline. .. 2.8 Confocal microscopyHEK293-SNAP-GLP-1R cells seeded on coverslips were labelled with SNAP-Surface 549 (New England Biolabs) prior to stimulation with the indicated agonist (100 nM) for 30 min, after which the cells were washed in PBS, fixed with 4% paraformaldehyde, mounted in Diamond Prolong mounting medium with DAPI, imaged with a Zeiss LSM780 inverted confocal microscope with a 63x/1.4 numerical aperture oil-immersion objective, and analysed in Fiji. ..

    FP Assay:

    Article Title: Evolution and Characterization of a Benzylguanine-binding RNA Aptamer
    Article Snippet: .. To test how well JX1 binds bG derivatives other than Atto488-bG we carried out the FP assay with a bG derivative containing a structurally and spectrally different fluorophore (Dy549-bG, SNAP-Surface 549® , New England Biolabs). .. We were able to observe a change in FP upon incubation of Dy549-bG with the JX1 aptamer; however, analysis of the binding data revealed a decrease in binding affinity (Kd = 1070 nM, ).

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    New England Biolabs fluorophore
    Cartoon of a bG-binding RNA aptamer and results from SELEX. (A) A bG-binding RNA aptamer can be used to target small molecule derivatives of bG to RNAs of interest. R represents a functional group such as a <t>fluorophore</t> or biotin. (B) Quantification of the fraction of RNA retained on bG agarose following each round of SELEX.
    Fluorophore, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorophore/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fluorophore - by Bioz Stars, 2021-03
    94/100 stars
      Buy from Supplier

    Image Search Results


    Cartoon of a bG-binding RNA aptamer and results from SELEX. (A) A bG-binding RNA aptamer can be used to target small molecule derivatives of bG to RNAs of interest. R represents a functional group such as a fluorophore or biotin. (B) Quantification of the fraction of RNA retained on bG agarose following each round of SELEX.

    Journal: Chemical communications (Cambridge, England)

    Article Title: Evolution and Characterization of a Benzylguanine-binding RNA Aptamer

    doi: 10.1039/c5cc07605f

    Figure Lengend Snippet: Cartoon of a bG-binding RNA aptamer and results from SELEX. (A) A bG-binding RNA aptamer can be used to target small molecule derivatives of bG to RNAs of interest. R represents a functional group such as a fluorophore or biotin. (B) Quantification of the fraction of RNA retained on bG agarose following each round of SELEX.

    Article Snippet: To test how well JX1 binds bG derivatives other than Atto488-bG we carried out the FP assay with a bG derivative containing a structurally and spectrally different fluorophore (Dy549-bG, SNAP-Surface 549® , New England Biolabs).

    Techniques: Binding Assay, Functional Assay

    FP competition assay for monitoring specificity of JX1 for bG. (A) Cartoon showing how an effective competitor of Atto488-bG for JX1 binding prevents binding of the RNA to the fluorophore results in low FP. (B) Results from the competition assay for selected guanine-containing molecules. High ΔFP values result from Atto488-bG binding to JX1 and lack of inhibition from the competitor. Each bar graph represents the average from three separate experiments and error bars represent ± S.D. N.D., Not Determined.

    Journal: Chemical communications (Cambridge, England)

    Article Title: Evolution and Characterization of a Benzylguanine-binding RNA Aptamer

    doi: 10.1039/c5cc07605f

    Figure Lengend Snippet: FP competition assay for monitoring specificity of JX1 for bG. (A) Cartoon showing how an effective competitor of Atto488-bG for JX1 binding prevents binding of the RNA to the fluorophore results in low FP. (B) Results from the competition assay for selected guanine-containing molecules. High ΔFP values result from Atto488-bG binding to JX1 and lack of inhibition from the competitor. Each bar graph represents the average from three separate experiments and error bars represent ± S.D. N.D., Not Determined.

    Article Snippet: To test how well JX1 binds bG derivatives other than Atto488-bG we carried out the FP assay with a bG derivative containing a structurally and spectrally different fluorophore (Dy549-bG, SNAP-Surface 549® , New England Biolabs).

    Techniques: Competitive Binding Assay, Binding Assay, Inhibition

    In vitro pharmacological properties of acylated biased GLP-1RAs. ( A ) Amino acid sequences in single letter code for the peptides used in this study, indicating the position of C16 diacid conjugation. ( B ) Equilibrium binding of each ligand in competition with exendin(9–39)-FITC in HEK293-SNAP-GLP-1R cells, n = 5. ( C ) Cyclic AMP (cAMP) production and β-arrestin-2 (βarr2) recruitment in CHO–K1-βarr2-EA-GLP-1R cells, 30-min incubation, n = 6, 4-parameter logistic fit of pooled data shown. ( D ) NanoBiT recruitment assays conducted in HEK293T cells transiently transfected with GLP-1R-SmBiT and miniG s -LgBiT (mG s , n = 10), miniG i -LgBiT (mG i , n = 4), miniG q -LgBiT (mG q , n = 4), or LgBiT-β-arrestin-2 (βarr2, n = 6). Vehicle response in light grey. Note the logarithmic scale for the y-axis. The heatmap shows AUC quantifications for each pathway normalised to the most efficacious ligand. ( E ) GLP-1R endocytosis measured in HEK293-SNAP-GLP-1R cells by DERET, indicated as AUC from kinetic traces shown in Supplementary Figure 1F , n = 5, 4-parameter logistic fit of pooled data shown. ( F ) Representative maximum intensity projection images, showing GLP-1R endocytosis in HEK293-SNAP-GLP-1R cells labelled with SNAP-Surface 549 prior to stimulation with indicated agonist (100 nM) for 30 min, n = 3, scale bar: 8 μm ∗p

    Journal: Molecular Metabolism

    Article Title: Disconnect between signalling potency and in vivo efficacy of pharmacokinetically optimised biased glucagon-like peptide-1 receptor agonists

    doi: 10.1016/j.molmet.2020.100991

    Figure Lengend Snippet: In vitro pharmacological properties of acylated biased GLP-1RAs. ( A ) Amino acid sequences in single letter code for the peptides used in this study, indicating the position of C16 diacid conjugation. ( B ) Equilibrium binding of each ligand in competition with exendin(9–39)-FITC in HEK293-SNAP-GLP-1R cells, n = 5. ( C ) Cyclic AMP (cAMP) production and β-arrestin-2 (βarr2) recruitment in CHO–K1-βarr2-EA-GLP-1R cells, 30-min incubation, n = 6, 4-parameter logistic fit of pooled data shown. ( D ) NanoBiT recruitment assays conducted in HEK293T cells transiently transfected with GLP-1R-SmBiT and miniG s -LgBiT (mG s , n = 10), miniG i -LgBiT (mG i , n = 4), miniG q -LgBiT (mG q , n = 4), or LgBiT-β-arrestin-2 (βarr2, n = 6). Vehicle response in light grey. Note the logarithmic scale for the y-axis. The heatmap shows AUC quantifications for each pathway normalised to the most efficacious ligand. ( E ) GLP-1R endocytosis measured in HEK293-SNAP-GLP-1R cells by DERET, indicated as AUC from kinetic traces shown in Supplementary Figure 1F , n = 5, 4-parameter logistic fit of pooled data shown. ( F ) Representative maximum intensity projection images, showing GLP-1R endocytosis in HEK293-SNAP-GLP-1R cells labelled with SNAP-Surface 549 prior to stimulation with indicated agonist (100 nM) for 30 min, n = 3, scale bar: 8 μm ∗p

    Article Snippet: 2.8 Confocal microscopyHEK293-SNAP-GLP-1R cells seeded on coverslips were labelled with SNAP-Surface 549 (New England Biolabs) prior to stimulation with the indicated agonist (100 nM) for 30 min, after which the cells were washed in PBS, fixed with 4% paraformaldehyde, mounted in Diamond Prolong mounting medium with DAPI, imaged with a Zeiss LSM780 inverted confocal microscope with a 63x/1.4 numerical aperture oil-immersion objective, and analysed in Fiji.

    Techniques: In Vitro, Conjugation Assay, Binding Assay, Incubation, Transfection

    Labeling efficiency of Rpb1 SNAP549 in nuclear extracts. Panels show SDS PAGE gels scanned for SNAP-Surface 549 fluorescence intensity. (A) Rpb1 SNAP549 /Spt5 DHFR yeast nuclear extract prepared with different concentrations of SNAP-Surface 549 dye and depleted of residual dye as described (see Materials and Methods). A concentration of 0.5 μM SNAP-Surface 549 was judged sufficient for maximal labeling. (B) Same as (A) but for Hek2 SNAP549 /Spt5 DHFR extract. Again, a concentration of 0.5 μM SNAP-Surface 549 was judged sufficient for maximal labeling. (C) Effectiveness of the dye depletion protocol at removing unincorporated dye after extract labeling. Lanes contain Rpb1 SNAP / Spt5 DHFR extract labeled with 0.5 μM SNAP-Surface 549 before (left) and after (right) dye depletion.

    Journal: bioRxiv

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

    doi: 10.1101/2020.06.01.127969

    Figure Lengend Snippet: Labeling efficiency of Rpb1 SNAP549 in nuclear extracts. Panels show SDS PAGE gels scanned for SNAP-Surface 549 fluorescence intensity. (A) Rpb1 SNAP549 /Spt5 DHFR yeast nuclear extract prepared with different concentrations of SNAP-Surface 549 dye and depleted of residual dye as described (see Materials and Methods). A concentration of 0.5 μM SNAP-Surface 549 was judged sufficient for maximal labeling. (B) Same as (A) but for Hek2 SNAP549 /Spt5 DHFR extract. Again, a concentration of 0.5 μM SNAP-Surface 549 was judged sufficient for maximal labeling. (C) Effectiveness of the dye depletion protocol at removing unincorporated dye after extract labeling. Lanes contain Rpb1 SNAP / Spt5 DHFR extract labeled with 0.5 μM SNAP-Surface 549 before (left) and after (right) dye depletion.

    Article Snippet: In-frame fusion protein expression and stability was confirmed by immunoblotting for the target protein (e.g., ), and in the case of SNAP fusions, by SDS-PAGE after labeling with SNAP-Surface 549 (New England Biolabs).

    Techniques: Labeling, SDS Page, Fluorescence, Concentration Assay

    Tagged yeast strains have growth rates, nuclear extract protein expression levels, and transcription activities similar to wild-type. (A) Western blot showing that the concentrations of Rpb1 SNAP in Rpb1 SNAP /Spt5 DHFR strain (YSB3403; see Table S5 ) (lane 2) and Rpb1 in parental strain (WT; YF702) are similar. TATA binding protein (TBP) was used as a loading control. Rpb1 was detected by monoclonal antibody 8WG16 ( 66 ) and TBP by a polyclonal anti-TBP antibody ( 67 ). (B) Western blot showing that the concentration of Spt5 DHFR in Rpb1 SNAP /Spt5 DHFR strain is similar to that of Spt5 in the parental strain. The anti-Spt5 antibody ( 68 ) was provided by Grant Hartzog. (C and D) RNA produced in bulk from plasmid transcription by Rpb1 SNAP /Spt5 DHFR (C) and Hek2 SNAP /Spt5 DHFR (D) nuclear extracts in comparison with extracts from corresponding parental strains (WT; see Table S5 ). Where indicated, extracts were labeled with 0.5 μM SNAP-Surface 549 and subsequently depleted of unreacted dye. Bulk in vitro transcription reactions were performed with 400 μM each ATP, CTP, and UTP supplemented with [α- 32 P]- UTP, separated by urea polyacrylamide gel electrophoresis, and visualized by autoradiography. The lower two bands in each lane are the transcripts from the two major transcription start sites of the template. The same volume of nuclear extract preparation was used in each reaction (10 μL in C and 8 μL in D); all extract preparations were 30-40 mg/mL protein. (E) Growth rates (mean ± S.E.; N = 5) of tagged strains relative to the corresponding parental (WT) strain. Colors denote correspondence between parental (YF702, blue; YF4, green) and tagged strains.

    Journal: bioRxiv

    Article Title: Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

    doi: 10.1101/2020.06.01.127969

    Figure Lengend Snippet: Tagged yeast strains have growth rates, nuclear extract protein expression levels, and transcription activities similar to wild-type. (A) Western blot showing that the concentrations of Rpb1 SNAP in Rpb1 SNAP /Spt5 DHFR strain (YSB3403; see Table S5 ) (lane 2) and Rpb1 in parental strain (WT; YF702) are similar. TATA binding protein (TBP) was used as a loading control. Rpb1 was detected by monoclonal antibody 8WG16 ( 66 ) and TBP by a polyclonal anti-TBP antibody ( 67 ). (B) Western blot showing that the concentration of Spt5 DHFR in Rpb1 SNAP /Spt5 DHFR strain is similar to that of Spt5 in the parental strain. The anti-Spt5 antibody ( 68 ) was provided by Grant Hartzog. (C and D) RNA produced in bulk from plasmid transcription by Rpb1 SNAP /Spt5 DHFR (C) and Hek2 SNAP /Spt5 DHFR (D) nuclear extracts in comparison with extracts from corresponding parental strains (WT; see Table S5 ). Where indicated, extracts were labeled with 0.5 μM SNAP-Surface 549 and subsequently depleted of unreacted dye. Bulk in vitro transcription reactions were performed with 400 μM each ATP, CTP, and UTP supplemented with [α- 32 P]- UTP, separated by urea polyacrylamide gel electrophoresis, and visualized by autoradiography. The lower two bands in each lane are the transcripts from the two major transcription start sites of the template. The same volume of nuclear extract preparation was used in each reaction (10 μL in C and 8 μL in D); all extract preparations were 30-40 mg/mL protein. (E) Growth rates (mean ± S.E.; N = 5) of tagged strains relative to the corresponding parental (WT) strain. Colors denote correspondence between parental (YF702, blue; YF4, green) and tagged strains.

    Article Snippet: In-frame fusion protein expression and stability was confirmed by immunoblotting for the target protein (e.g., ), and in the case of SNAP fusions, by SDS-PAGE after labeling with SNAP-Surface 549 (New England Biolabs).

    Techniques: Expressing, Western Blot, Binding Assay, Concentration Assay, Produced, Plasmid Preparation, Labeling, In Vitro, Polyacrylamide Gel Electrophoresis, Autoradiography

    Counting statistics for the number of steps observed during loss of fluorescence from either U1-double DHFR or BBP-SNAP f binding events in three-color CoSMoS experiments. ( A ) Number of steps observed during loss of U1 fluorescence. Each U1 molecule contains two fluorophores, which can result in either 1 or two steps. 91% of U1 binding events are consistent with presence of only a single U1 molecule on the pre-mRNA. ( B ) Number of steps observed during loss of BBP fluorescence. Each BBP molecule contains a single fluorophore. 88% of BBP binding events are consistent with presence of only a single BBP molecule on the pre-mRNA.

    Journal: eLife

    Article Title: Dynamics and consequences of spliceosome E complex formation

    doi: 10.7554/eLife.27592

    Figure Lengend Snippet: Counting statistics for the number of steps observed during loss of fluorescence from either U1-double DHFR or BBP-SNAP f binding events in three-color CoSMoS experiments. ( A ) Number of steps observed during loss of U1 fluorescence. Each U1 molecule contains two fluorophores, which can result in either 1 or two steps. 91% of U1 binding events are consistent with presence of only a single U1 molecule on the pre-mRNA. ( B ) Number of steps observed during loss of BBP fluorescence. Each BBP molecule contains a single fluorophore. 88% of BBP binding events are consistent with presence of only a single BBP molecule on the pre-mRNA.

    Article Snippet: SNAPf -tagged proteins were labeled by incubation of the lysate for 30 min at room temperature with the fluorophore (e.g., benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs) before gel filtration.

    Techniques: Fluorescence, Binding Assay

    Single molecule analysis of BBP-SNAP f binding dynamics. ( A ) Cartoon of a two-color CoSMoS experiment for observing BBP binding dynamics. BBP was labeled with a single green-excited fluorophore while RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Graphic representation of capped RNAs with variable BS and ΨBS used in these experiments and their corresponding label number. RNA sequences are given in Supplementary file 1 . ( C ) Bar graph comparing the relative number of BBP binding events observed on each RNA depicted in panel ( B ). ( D ) Rastergram depicting BBP binding events on RNAs containing both a BS and ΨBS (RNA 3). ( E ) Rastergram depicting BBP binding events on RNAs containing only the BS (RNA 9). ( F ) Rastergram depicting BBP binding events on RNAs containing only the ΨBS (RNA 10). ( G ) Probability density histogram of dwell times for BBP on RNAs with variable BS and ΨBS. Lines represent fits of the distributions of dwell times to equations containing one or two exponential terms. ( H–J ) Bar graph comparison of the fit parameters (τ 1 , panel H; τ 2 , panel I; the τ 2 amplitude A 2 , panel J) obtained from analysis of the dwell time distributions of BBP binding events on RNAs 3, 9, and 10. Details of the fit parameters for data shown in ( H–J ) can be found in Supplementary file 3 . Error bars in ( C, G ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( H–J ) represent the fit parameters ± S.D.

    Journal: eLife

    Article Title: Dynamics and consequences of spliceosome E complex formation

    doi: 10.7554/eLife.27592

    Figure Lengend Snippet: Single molecule analysis of BBP-SNAP f binding dynamics. ( A ) Cartoon of a two-color CoSMoS experiment for observing BBP binding dynamics. BBP was labeled with a single green-excited fluorophore while RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Graphic representation of capped RNAs with variable BS and ΨBS used in these experiments and their corresponding label number. RNA sequences are given in Supplementary file 1 . ( C ) Bar graph comparing the relative number of BBP binding events observed on each RNA depicted in panel ( B ). ( D ) Rastergram depicting BBP binding events on RNAs containing both a BS and ΨBS (RNA 3). ( E ) Rastergram depicting BBP binding events on RNAs containing only the BS (RNA 9). ( F ) Rastergram depicting BBP binding events on RNAs containing only the ΨBS (RNA 10). ( G ) Probability density histogram of dwell times for BBP on RNAs with variable BS and ΨBS. Lines represent fits of the distributions of dwell times to equations containing one or two exponential terms. ( H–J ) Bar graph comparison of the fit parameters (τ 1 , panel H; τ 2 , panel I; the τ 2 amplitude A 2 , panel J) obtained from analysis of the dwell time distributions of BBP binding events on RNAs 3, 9, and 10. Details of the fit parameters for data shown in ( H–J ) can be found in Supplementary file 3 . Error bars in ( C, G ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( H–J ) represent the fit parameters ± S.D.

    Article Snippet: SNAPf -tagged proteins were labeled by incubation of the lysate for 30 min at room temperature with the fluorophore (e.g., benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs) before gel filtration.

    Techniques: Binding Assay, Labeling

    Single molecule analysis of U1 binding after ablation of the 5' end of the snRNA. ( A ) Cartoon of a two-color CoSMoS experiment for observing U1 binding dynamics. U1 was labeled with two green-excited fluorophores while the RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Confirmation of U1 ablation by primer extension. Primer extension of the U2 snRNA is included as a loading control. Quantification of the band intensities indicate that ≥96% of the U1 snRNA was cleaved by RNase H. ( C ) Bar graph comparing the relative number of U1 binding events observed on RNAs 3 or 7, in the presence or absence of CA and/or the RNase H ablation oligo. ( D–F ) Bar graph comparison of the fit parameters (τ 1 , panel D; τ 2 , panel E; the τ 2 amplitude A 2 , panel F) obtained from analysis of the dwell time distributions of U1 binding events. Details of the fit parameters for data shown in ( D–F ) can be found in Supplementary file 2 . Error bars in ( C ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( D–F ) represent the fit parameters ± S.D.

    Journal: eLife

    Article Title: Dynamics and consequences of spliceosome E complex formation

    doi: 10.7554/eLife.27592

    Figure Lengend Snippet: Single molecule analysis of U1 binding after ablation of the 5' end of the snRNA. ( A ) Cartoon of a two-color CoSMoS experiment for observing U1 binding dynamics. U1 was labeled with two green-excited fluorophores while the RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Confirmation of U1 ablation by primer extension. Primer extension of the U2 snRNA is included as a loading control. Quantification of the band intensities indicate that ≥96% of the U1 snRNA was cleaved by RNase H. ( C ) Bar graph comparing the relative number of U1 binding events observed on RNAs 3 or 7, in the presence or absence of CA and/or the RNase H ablation oligo. ( D–F ) Bar graph comparison of the fit parameters (τ 1 , panel D; τ 2 , panel E; the τ 2 amplitude A 2 , panel F) obtained from analysis of the dwell time distributions of U1 binding events. Details of the fit parameters for data shown in ( D–F ) can be found in Supplementary file 2 . Error bars in ( C ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( D–F ) represent the fit parameters ± S.D.

    Article Snippet: SNAPf -tagged proteins were labeled by incubation of the lysate for 30 min at room temperature with the fluorophore (e.g., benzylguanine-Dy549/SNAP-Surface 549, New England Biolabs) before gel filtration.

    Techniques: Binding Assay, Labeling