alexa fluor 488  (New England Biolabs)


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    Name:
    SNAP Surface Alexa Fluor 488
    Description:
    SNAP Surface Alexa Fluor 488 50 nmol
    Catalog Number:
    s9129s
    Price:
    380
    Size:
    50 nmol
    Category:
    Fluorochromes
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    Structured Review

    New England Biolabs alexa fluor 488
    SNAP Surface Alexa Fluor 488
    SNAP Surface Alexa Fluor 488 50 nmol
    https://www.bioz.com/result/alexa fluor 488/product/New England Biolabs
    Average 95 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    alexa fluor 488 - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Emerin induces nuclear breakage in Xenopus extract and early embryos"

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E18-05-0277

    TRC40 rescues emerin’s nuclear localization. (A) Experiments were performed as in Figure 1 , except that the extract was supplemented with 1 µM DiI and 0.4 µM SNAP-emerin conjugated to Alexa Fluor 488. Representative images of small and large emerin puncta are shown. (B) Nuclei assembled in X. laevis egg extract as shown in Figure 1 were supplemented with 8 nM TRC40-EMD or an equivalent volume of dialysis buffer. After a 30-min incubation, nuclei were fixed, spun down onto coverslips, and stained with an anti-emerin antibody and Hoechst. Emerin images were acquired with the same exposure time. Representative images are shown.
    Figure Legend Snippet: TRC40 rescues emerin’s nuclear localization. (A) Experiments were performed as in Figure 1 , except that the extract was supplemented with 1 µM DiI and 0.4 µM SNAP-emerin conjugated to Alexa Fluor 488. Representative images of small and large emerin puncta are shown. (B) Nuclei assembled in X. laevis egg extract as shown in Figure 1 were supplemented with 8 nM TRC40-EMD or an equivalent volume of dialysis buffer. After a 30-min incubation, nuclei were fixed, spun down onto coverslips, and stained with an anti-emerin antibody and Hoechst. Emerin images were acquired with the same exposure time. Representative images are shown.

    Techniques Used: Incubation, Staining

    2) Product Images from "Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy"

    Article Title: Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.08.074

    Functional Characterization of the hu-sCD4-SNAP-tag Fusion Protein (A) Western blot showing the expression of SNAP-tagged sCD4 (sCD4-SNAP) in the supernatant from the 293FS-transfected cells. Bands corresponding to sCD4-SNAP fusion protein and untagged sCD4 were detected using anti-huCD4 antibody. L, molecular weight ladder; W, wash; E, elute; FT, flow through; H, harvest; sCD4, positive control at 75 and 150 ng concentration. (B) SDS-PAGE with Coomassie staining showing successful purification of sCD4-SNAP from transfected 293FS cells using CNBr-activated Sepharose conjugated with an anti-CD4 antibody. Samples are as described in (A). (C) FCS autocorrelation plots for sCD4-SNAP-A488 alone and in complex with monomeric gp120, SOSIP.664 (BG505), and SOSIP.664.D7 (BG505.D7) trimers are shown. (D) FCS binding of Alexa 647 labeled mAbs b12 and 17b to HIV-1 BaL virions with or without 100 μg/mL sCD4-SNAP. Data are presented as the mean of three experiments ± SEM. ***Average percentage binding is significantly (p
    Figure Legend Snippet: Functional Characterization of the hu-sCD4-SNAP-tag Fusion Protein (A) Western blot showing the expression of SNAP-tagged sCD4 (sCD4-SNAP) in the supernatant from the 293FS-transfected cells. Bands corresponding to sCD4-SNAP fusion protein and untagged sCD4 were detected using anti-huCD4 antibody. L, molecular weight ladder; W, wash; E, elute; FT, flow through; H, harvest; sCD4, positive control at 75 and 150 ng concentration. (B) SDS-PAGE with Coomassie staining showing successful purification of sCD4-SNAP from transfected 293FS cells using CNBr-activated Sepharose conjugated with an anti-CD4 antibody. Samples are as described in (A). (C) FCS autocorrelation plots for sCD4-SNAP-A488 alone and in complex with monomeric gp120, SOSIP.664 (BG505), and SOSIP.664.D7 (BG505.D7) trimers are shown. (D) FCS binding of Alexa 647 labeled mAbs b12 and 17b to HIV-1 BaL virions with or without 100 μg/mL sCD4-SNAP. Data are presented as the mean of three experiments ± SEM. ***Average percentage binding is significantly (p

    Techniques Used: Functional Assay, Western Blot, Expressing, Transfection, Molecular Weight, Flow Cytometry, Positive Control, Concentration Assay, SDS Page, Staining, Purification, Binding Assay, Labeling

    3) Product Images from "Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen"

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0249-x

    Binding analysis of recombinant TTC and TTC-FITC on isolated PBMCs. Surface staining of CD27 + memory B cells ( a ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( c ) using recombinant TTC (10 nM) and anti-His5 Alexa Fluor 488 antibody (1:100). Surface staining of CD27 + memory B cells ( b ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( d ) using a FITC-coupled TTC peptide (1:25)
    Figure Legend Snippet: Binding analysis of recombinant TTC and TTC-FITC on isolated PBMCs. Surface staining of CD27 + memory B cells ( a ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( c ) using recombinant TTC (10 nM) and anti-His5 Alexa Fluor 488 antibody (1:100). Surface staining of CD27 + memory B cells ( b ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( d ) using a FITC-coupled TTC peptide (1:25)

    Techniques Used: Binding Assay, Recombinant, Isolation, Staining

    Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein
    Figure Legend Snippet: Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein

    Techniques Used: Labeling, SDS Page, Fluorescence, In Vivo Imaging, Staining, SDS-Gel, Marker

    Dose-dependent binding analysis of the recombinant fusion protein TTC-ETA’ on hybridoma cells. Various concentrations (1–400 nM) of TTC-ETA’ were used to determine a dose-dependent binding activity on TTC-reactive hybridoma cell line 5E4 ( a ) and to exclude specific binding to the control hybridoma cell line 8.18-C5 ( b ). The detection of bound protein was carried out by flow cytometry using a Penta-His Alexa Fluor 488 Conjugate antibody. Measurements were performed in triplicates (n = 3); error bars indicate SD. The recombinant TTC-ETA’ exhibits a dosedependent binding on the target hybridoma cell line 5E4, whereas no binding could be determined on the control hybridoma cell line 8.18-C5
    Figure Legend Snippet: Dose-dependent binding analysis of the recombinant fusion protein TTC-ETA’ on hybridoma cells. Various concentrations (1–400 nM) of TTC-ETA’ were used to determine a dose-dependent binding activity on TTC-reactive hybridoma cell line 5E4 ( a ) and to exclude specific binding to the control hybridoma cell line 8.18-C5 ( b ). The detection of bound protein was carried out by flow cytometry using a Penta-His Alexa Fluor 488 Conjugate antibody. Measurements were performed in triplicates (n = 3); error bars indicate SD. The recombinant TTC-ETA’ exhibits a dosedependent binding on the target hybridoma cell line 5E4, whereas no binding could be determined on the control hybridoma cell line 8.18-C5

    Techniques Used: Binding Assay, Recombinant, Activity Assay, Flow Cytometry, Cytometry

    Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )
    Figure Legend Snippet: Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )

    Techniques Used: Binding Assay, Recombinant, Staining, Fluorescence

    4) Product Images from "Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro"

    Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

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

    doi: 10.1073/pnas.1619473114

    Mlph’s phosphorylation state does not interfere substantially with actin binding. ( A ) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left ) and the phosphorylated (Phos; Right ) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. ( B ) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)
    Figure Legend Snippet: Mlph’s phosphorylation state does not interfere substantially with actin binding. ( A ) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left ) and the phosphorylated (Phos; Right ) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. ( B ) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)

    Techniques Used: Binding Assay, Labeling, Incubation, Fluorescence

    Related Articles

    Transduction:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C. .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C.

    Clone Assay:

    Article Title: Protein dynamics during presynaptic complex assembly on individual ssDNA molecules
    Article Snippet: SNAP–Rad52 was labeled with SNAP–Surface Alexa Fluor 488 (ε495 nm =71,000 cm−1 M−1 ) or SNAP–Surface Alexa Fluor 546 (ε556 nm =104,000 cm−1 M−1 ) overnight at 4°C, as per the manufacturers instructions (NEB). .. Rad52 (codons 34–504)was cloned with an N–terminal 6xHis and a C–terminal intein/chitin–binding domain, and purified from bacteria as described above for the SNAP–tagged proteins.

    Filtration:

    Article Title: Single-molecule visualization of fast polymerase turnover in the bacterial replisome
    Article Snippet: Fluorescent labeling of SNAP-α Two different fluorescent probes, SNAP-Surface 649 (red) and SNAP-Surface Alexa Fluor 488 (green; New England Biolabs), were used to label SNAP-α. .. Following the coupling, the reaction mixture was supplemented with 1 mM EDTA and excess dye was removed by gel filtration at 1 ml/min through a column (1.5 × 10 cm) of Sephadex G-25 (GE Healthcare, Chicago, IL) in buffer Fα+1 mM EDTA.

    Stable Transfection:

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: Flow cytometry HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Cytometry:

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: Paragraph title: Flow cytometry ... The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins
    Article Snippet: Paragraph title: Flow Cytometry ... For SNAP tag staining we used SNAP surface Alexa fluor 488 (NEB).

    Construct:

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: Flow cytometry HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: Protein dynamics during presynaptic complex assembly on individual ssDNA molecules
    Article Snippet: Use of this longer reading frame provides an additional 34 amino acids placed between the SNAP tag and the N–terminus of Rad52; we refer to this construct as SNAP–34–Rad52. .. SNAP–Rad52 was labeled with SNAP–Surface Alexa Fluor 488 (ε495 nm =71,000 cm−1 M−1 ) or SNAP–Surface Alexa Fluor 546 (ε556 nm =104,000 cm−1 M−1 ) overnight at 4°C, as per the manufacturers instructions (NEB).

    Incubation:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C. .. The fluorophore labeled probe was then dialyzed overnight in 1× PBS containing 1 mM DTT to remove unreacted substrates.

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: .. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents. .. Fluorescent images were obtained using confocal laser scanning microscopes (LSM5 Pascal; Carl Zeiss, Jena, Germany; FluoView FV10i, Olympus).

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C. .. The plate was centrifuged twice, cells were resuspended in ice-cold 0.5% BSA/PBS, and assayed immediately on an ADP Cyan flow cytometer.

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Briefly, 1 μg SNAP-TTC protein was mixed with 2 nmol BG-647 or BG-488 solution prepared from a 50 nmol stock and incubated for 1 h at room temperature.

    Article Title: Caenorhabditis elegans Galectins LEC-6 and LEC-10 Interact with Similar Glycoconjugates in the Intestine
    Article Snippet: Labeling of SNAP-galectin Purified SNAP-galectin protein at a concentration of 5 μm was incubated with 10 μm substrate flurophore in phosphate buffer supplemented with 1 mm DTT for 1 h at 37 °C. .. Fluorophores used for labeling were SNAP-surface Alexa Fluor 488 (NEB S9129S), SNAP-surface Alexa Fluor 546 (NEB S9132S) and SNAP-surface-IR800.

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C. .. The plate was centrifuged twice, cells were resuspended in ice-cold 0.5% BSA/PBS, and assayed immediately on an ADP Cyan flow cytometer.

    Expressing:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: 6× His-SNAP-CBD expression was induced using 1 mM IPTG and purified by affinity chromatography using Nickel resin. .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C.

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: In some experiments, pSNAPf -ADRβ2 (New England Biolabs, Ipswich, MA, USA), an expression plasmid for β2 adrenergic receptor fused to SNAPf , was cotransfected into HEK-293 cells. .. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents.

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: Flow cytometry HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Transformation Assay:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: Antibodies used for immunostaining The chitin binding probe was prepared in our laboratory. pYZ205, a 6× His-SNAP-CBD plasmid [provided by New England Biolabs (NEB) upon request], was transformed to an E.coli strain. .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C.

    Transfection:

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: After transfection with pcDNAHOXTR-376R, pcDNAHOXTR-376G, or pcDNAHOXTR-376C, the cells were incubated at 37°C in serum-free DMEM for different times, in the absence or presence of OXT (100 nM). .. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents.

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins
    Article Snippet: Briefly, 2-5 x 104 transfected cells were placed in each well of a 96 well V bottom plate and stained with saturating amounts of fluorescently labeled monoclonal antibodies (FITC or phycoerythrin (PE)) Anti HA (Miltenyi Biotec); PE CD20, and PE Anti DARC (R & D Systems); PE Anti CXCR4 (Biolegend); PE anti-CD24 (BD Bioscience), or PE labeled isotype controls (Santa Cruz Biotechnology). .. For SNAP tag staining we used SNAP surface Alexa fluor 488 (NEB).

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: Flow cytometry HEK293 cells transiently transfected with SNAP-tagged FZD constructs and mG constructs or stably expressing SNAP-FZD6 and SNAP-FZD6 R416A6.32 were grown in a 6-well plate. .. The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Cell Culture:

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: Measurement of receptor recycling HEK-293 cells were plated on poly-D -ornithine-coated glass coverslips and cultured overnight. .. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents.

    Generated:

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos
    Article Snippet: .. SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB). .. The TRC40-EMD complex was purified as previously described ( ).

    other:

    Article Title: Intracellular Delivery of Bioactive Molecules using Light-Addressable Nanocapsules
    Article Snippet: AGT-PP1 and SNAP-Surface™ Alexa Fluor® 488 were all purchased from New England BioLabs (Ipswich, MA).

    Imaging:

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Samples were taken to determine the coupling efficiency by SDS-PAGE and the fluorescence signal was detected using the CRi Maestro imaging system with appropriate filter sets (Perkin Elmer, Waltham, MA, USA).

    Sonication:

    Article Title: Protein dynamics during presynaptic complex assembly on individual ssDNA molecules
    Article Snippet: Bacteria (E. coli Rossetta™ ) were grown at 37°C, induced overnight at 18°C with 0.5 mM IPTG, and lysed by sonication. .. SNAP–Rad52 was labeled with SNAP–Surface Alexa Fluor 488 (ε495 nm =71,000 cm−1 M−1 ) or SNAP–Surface Alexa Fluor 546 (ε556 nm =104,000 cm−1 M−1 ) overnight at 4°C, as per the manufacturers instructions (NEB).

    Binding Assay:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C. .. The fluorophore labeled probe was then dialyzed overnight in 1× PBS containing 1 mM DTT to remove unreacted substrates.

    Fluorescence:

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C. .. The median fluorescence intensity (MFI) data were analyzed using FlowJo V10 (Tree Star).

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Samples were taken to determine the coupling efficiency by SDS-PAGE and the fluorescence signal was detected using the CRi Maestro imaging system with appropriate filter sets (Perkin Elmer, Waltham, MA, USA).

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C. .. The median fluorescence intensity (MFI) data were analyzed using FlowJo V10 (Tree Star).

    Flow Cytometry:

    Article Title: A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection
    Article Snippet: Paragraph title: Flow cytometry ... The plate was then centrifuged at 400× g for 5 min and subsequently cells were incubated with SNAP-substrate: either SNAP-Surface Alexa Fluor 488 (NEB #S9129S), SNAP-Surface Alexa Fluor 647 (NEB #S9136S) or SNAP-Cell 647-SiR (NEB #S9102S) at 1:200 dilution in complete DMEM medium for 30 min at 37 °C.

    Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins
    Article Snippet: Paragraph title: Flow Cytometry ... For SNAP tag staining we used SNAP surface Alexa fluor 488 (NEB).

    Labeling:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C. .. The fluorophore labeled probe was then dialyzed overnight in 1× PBS containing 1 mM DTT to remove unreacted substrates.

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: .. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents. .. Fluorescent images were obtained using confocal laser scanning microscopes (LSM5 Pascal; Carl Zeiss, Jena, Germany; FluoView FV10i, Olympus).

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos
    Article Snippet: .. SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB). .. The TRC40-EMD complex was purified as previously described ( ).

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: .. SNAPf -tagged β2 adrenergic receptor was labeled with SNAP-Surface Alexa Fluor 488 (SNAPf -ADRβ2, green). .. DAPI was used to stain cell nuclei (DAPI, blue).

    Article Title: Single-molecule visualization of fast polymerase turnover in the bacterial replisome
    Article Snippet: .. Fluorescent labeling of SNAP-α Two different fluorescent probes, SNAP-Surface 649 (red) and SNAP-Surface Alexa Fluor 488 (green; New England Biolabs), were used to label SNAP-α. .. All labeling reactions were carried out using a twofold molar excess of dye with 27 μM SNAP-α in 1 ml of 50 mM Tris-HCl pH 7.6, 2 mM dithiothreitol, 100 mM NaCl, 5% (v /v ) glycerol (buffer Fα) for 2 hr at 23°C, followed by 6°C overnight with gentle rotation.

    Article Title: Caenorhabditis elegans Galectins LEC-6 and LEC-10 Interact with Similar Glycoconjugates in the Intestine
    Article Snippet: .. Fluorophores used for labeling were SNAP-surface Alexa Fluor 488 (NEB S9129S), SNAP-surface Alexa Fluor 546 (NEB S9132S) and SNAP-surface-IR800. .. After incubation, each labeled protein was dialyzed overnight in phosphate buffer containing 1 mm DTT to remove nonreacted substrates.

    Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins
    Article Snippet: Briefly, 2-5 x 104 transfected cells were placed in each well of a 96 well V bottom plate and stained with saturating amounts of fluorescently labeled monoclonal antibodies (FITC or phycoerythrin (PE)) Anti HA (Miltenyi Biotec); PE CD20, and PE Anti DARC (R & D Systems); PE Anti CXCR4 (Biolegend); PE anti-CD24 (BD Bioscience), or PE labeled isotype controls (Santa Cruz Biotechnology). .. For SNAP tag staining we used SNAP surface Alexa fluor 488 (NEB).

    Article Title: Protein dynamics during presynaptic complex assembly on individual ssDNA molecules
    Article Snippet: .. SNAP–Rad52 was labeled with SNAP–Surface Alexa Fluor 488 (ε495 nm =71,000 cm−1 M−1 ) or SNAP–Surface Alexa Fluor 546 (ε556 nm =104,000 cm−1 M−1 ) overnight at 4°C, as per the manufacturers instructions (NEB). .. Unreacted dye was removed with a Sephadex G20 spin column, and labeling efficiency (~0.55–0.60 dyes per Rad52 monomer) was determined by comparing protein UV absorbance to the absorbance of the Alexa dye.

    Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro
    Article Snippet: Paragraph title: Fluorescent Labeling of Proteins. ... Before the elution of the protein from the Ni-NTA beads, the wash buffer was supplemented with 20 µM SNAP-tag substrate SNAP-Surface Alexa Fluor 647 or SNAP-Surface Alexa Fluor 488 (New England Biolabs) and was added to the Ni-NTA beads.

    Purification:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C. .. The fluorophore labeled probe was then dialyzed overnight in 1× PBS containing 1 mM DTT to remove unreacted substrates.

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos
    Article Snippet: Emerin purified using each of these different schemes induced similar frequencies of nuclear breakage in extract. .. SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB).

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: .. Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Briefly, 1 μg SNAP-TTC protein was mixed with 2 nmol BG-647 or BG-488 solution prepared from a 50 nmol stock and incubated for 1 h at room temperature.

    Article Title: Caenorhabditis elegans Galectins LEC-6 and LEC-10 Interact with Similar Glycoconjugates in the Intestine
    Article Snippet: Labeling of SNAP-galectin Purified SNAP-galectin protein at a concentration of 5 μm was incubated with 10 μm substrate flurophore in phosphate buffer supplemented with 1 mm DTT for 1 h at 37 °C. .. Fluorophores used for labeling were SNAP-surface Alexa Fluor 488 (NEB S9129S), SNAP-surface Alexa Fluor 546 (NEB S9132S) and SNAP-surface-IR800.

    Article Title: Protein dynamics during presynaptic complex assembly on individual ssDNA molecules
    Article Snippet: The clarified lysate was purified over a chitin column (NEB), washed with buffer A (50 mM Tris–HCl [pH 7.5], 600 mM NaCl) plus 1 mM EDTA, and eluted overnight in buffer A plus 1 mM EDTA and 50 mM DTT. .. SNAP–Rad52 was labeled with SNAP–Surface Alexa Fluor 488 (ε495 nm =71,000 cm−1 M−1 ) or SNAP–Surface Alexa Fluor 546 (ε556 nm =104,000 cm−1 M−1 ) overnight at 4°C, as per the manufacturers instructions (NEB).

    Protein Purification:

    Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro
    Article Snippet: The SNAP-tagged Rab27a was labeled during the protein purification. .. Before the elution of the protein from the Ni-NTA beads, the wash buffer was supplemented with 20 µM SNAP-tag substrate SNAP-Surface Alexa Fluor 647 or SNAP-Surface Alexa Fluor 488 (New England Biolabs) and was added to the Ni-NTA beads.

    Immunostaining:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: Paragraph title: Antibodies used for immunostaining ... To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C.

    SDS Page:

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos
    Article Snippet: To approximate protein concentrations, emerin stock solutions and BSA standards were separated on SDS–PAGE gels and stained with Coomassie. .. SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB).

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Samples were taken to determine the coupling efficiency by SDS-PAGE and the fluorescence signal was detected using the CRi Maestro imaging system with appropriate filter sets (Perkin Elmer, Waltham, MA, USA).

    Plasmid Preparation:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: Antibodies used for immunostaining The chitin binding probe was prepared in our laboratory. pYZ205, a 6× His-SNAP-CBD plasmid [provided by New England Biolabs (NEB) upon request], was transformed to an E.coli strain. .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C.

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: In some experiments, pSNAPf -ADRβ2 (New England Biolabs, Ipswich, MA, USA), an expression plasmid for β2 adrenergic receptor fused to SNAPf , was cotransfected into HEK-293 cells. .. The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents.

    Software:

    Article Title: Non-synonymous single-nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis
    Article Snippet: The cells were labeled with SNAP-Surface Alexa Fluor 488 (2 μM; New England Biolabs), fixed with 4% paraformaldehyde, and reacted with goat anti-OXTR antibody (N-19; 1:200; Santa Cruz) at 4°C overnight, followed by incubation with donkey anti-goat IgG (H + L) antibody conjugated with Alexa Fluor 594 (1:500; Invitrogen); none of the solutions contained any cell-permeabilizing reagents. .. Data were analyzed using the FV10-ASW software (Olympus).

    Affinity Chromatography:

    Article Title: rebuff regulates apical luminal matrix to control tube size in Drosophila trachea
    Article Snippet: 6× His-SNAP-CBD expression was induced using 1 mM IPTG and purified by affinity chromatography using Nickel resin. .. To label the purified chitin binding probe with SNAP tag, SNAP-surface Alexa Fluor 488 (NEB; cat# S9129S) was added and incubated for 1 h at 37°C.

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos
    Article Snippet: In other instances, dialyzed emerin was subjected to Ni-NTA affinity chromatography and an additional round of dialysis. .. SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB).

    Concentration Assay:

    Article Title: Caenorhabditis elegans Galectins LEC-6 and LEC-10 Interact with Similar Glycoconjugates in the Intestine
    Article Snippet: Labeling of SNAP-galectin Purified SNAP-galectin protein at a concentration of 5 μm was incubated with 10 μm substrate flurophore in phosphate buffer supplemented with 1 mm DTT for 1 h at 37 °C. .. Fluorophores used for labeling were SNAP-surface Alexa Fluor 488 (NEB S9129S), SNAP-surface Alexa Fluor 546 (NEB S9132S) and SNAP-surface-IR800.

    Staining:

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos
    Article Snippet: To approximate protein concentrations, emerin stock solutions and BSA standards were separated on SDS–PAGE gels and stained with Coomassie. .. SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB).

    Article Title: Snorkel: An Epitope Tagging System for Measuring the Surface Expression of Membrane Proteins
    Article Snippet: .. For SNAP tag staining we used SNAP surface Alexa fluor 488 (NEB). .. All staining was in a final volume of 50 µl of 10% normal goat serum (heat inactivated, 30 minutes at 56o C) in PBS with 0.025% sodium azide and was performed at 2-8o C. After 30 minutes with gentle shaking cells were washed three times with cold 1% bovine serum albumin (BSA) in PBS with 0.025% sodium azide and analyzed.

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    New England Biolabs alexa fluor 488
    TRC40 rescues emerin’s nuclear localization. (A) Experiments were performed as in Figure 1 , except that the extract was supplemented with 1 µM DiI and 0.4 µM SNAP-emerin conjugated to <t>Alexa</t> Fluor 488. Representative images of small and large emerin puncta are shown. (B) Nuclei assembled in X. laevis egg extract as shown in Figure 1 were supplemented with 8 nM TRC40-EMD or an equivalent volume of dialysis buffer. After a 30-min incubation, nuclei were fixed, spun down onto coverslips, and stained with an anti-emerin antibody and Hoechst. Emerin images were acquired with the same exposure time. Representative images are shown.
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    TRC40 rescues emerin’s nuclear localization. (A) Experiments were performed as in Figure 1 , except that the extract was supplemented with 1 µM DiI and 0.4 µM SNAP-emerin conjugated to Alexa Fluor 488. Representative images of small and large emerin puncta are shown. (B) Nuclei assembled in X. laevis egg extract as shown in Figure 1 were supplemented with 8 nM TRC40-EMD or an equivalent volume of dialysis buffer. After a 30-min incubation, nuclei were fixed, spun down onto coverslips, and stained with an anti-emerin antibody and Hoechst. Emerin images were acquired with the same exposure time. Representative images are shown.

    Journal: Molecular Biology of the Cell

    Article Title: Emerin induces nuclear breakage in Xenopus extract and early embryos

    doi: 10.1091/mbc.E18-05-0277

    Figure Lengend Snippet: TRC40 rescues emerin’s nuclear localization. (A) Experiments were performed as in Figure 1 , except that the extract was supplemented with 1 µM DiI and 0.4 µM SNAP-emerin conjugated to Alexa Fluor 488. Representative images of small and large emerin puncta are shown. (B) Nuclei assembled in X. laevis egg extract as shown in Figure 1 were supplemented with 8 nM TRC40-EMD or an equivalent volume of dialysis buffer. After a 30-min incubation, nuclei were fixed, spun down onto coverslips, and stained with an anti-emerin antibody and Hoechst. Emerin images were acquired with the same exposure time. Representative images are shown.

    Article Snippet: SNAP-emerin generated from pDL97 was labeled with Janelia Fluor 549 ( ) or Alexa Fluor 488 according to the manufacturer’s protocol (S9129, NEB).

    Techniques: Incubation, Staining

    Functional Characterization of the hu-sCD4-SNAP-tag Fusion Protein (A) Western blot showing the expression of SNAP-tagged sCD4 (sCD4-SNAP) in the supernatant from the 293FS-transfected cells. Bands corresponding to sCD4-SNAP fusion protein and untagged sCD4 were detected using anti-huCD4 antibody. L, molecular weight ladder; W, wash; E, elute; FT, flow through; H, harvest; sCD4, positive control at 75 and 150 ng concentration. (B) SDS-PAGE with Coomassie staining showing successful purification of sCD4-SNAP from transfected 293FS cells using CNBr-activated Sepharose conjugated with an anti-CD4 antibody. Samples are as described in (A). (C) FCS autocorrelation plots for sCD4-SNAP-A488 alone and in complex with monomeric gp120, SOSIP.664 (BG505), and SOSIP.664.D7 (BG505.D7) trimers are shown. (D) FCS binding of Alexa 647 labeled mAbs b12 and 17b to HIV-1 BaL virions with or without 100 μg/mL sCD4-SNAP. Data are presented as the mean of three experiments ± SEM. ***Average percentage binding is significantly (p

    Journal: Cell reports

    Article Title: Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy

    doi: 10.1016/j.celrep.2019.08.074

    Figure Lengend Snippet: Functional Characterization of the hu-sCD4-SNAP-tag Fusion Protein (A) Western blot showing the expression of SNAP-tagged sCD4 (sCD4-SNAP) in the supernatant from the 293FS-transfected cells. Bands corresponding to sCD4-SNAP fusion protein and untagged sCD4 were detected using anti-huCD4 antibody. L, molecular weight ladder; W, wash; E, elute; FT, flow through; H, harvest; sCD4, positive control at 75 and 150 ng concentration. (B) SDS-PAGE with Coomassie staining showing successful purification of sCD4-SNAP from transfected 293FS cells using CNBr-activated Sepharose conjugated with an anti-CD4 antibody. Samples are as described in (A). (C) FCS autocorrelation plots for sCD4-SNAP-A488 alone and in complex with monomeric gp120, SOSIP.664 (BG505), and SOSIP.664.D7 (BG505.D7) trimers are shown. (D) FCS binding of Alexa 647 labeled mAbs b12 and 17b to HIV-1 BaL virions with or without 100 μg/mL sCD4-SNAP. Data are presented as the mean of three experiments ± SEM. ***Average percentage binding is significantly (p

    Article Snippet: 100 μM of sCD4-SNAP was then fluorescently labeled with a SNAP surface Alexa Fluor 488 labeling kit (NEB) and dialyzed against PBS as necessary.

    Techniques: Functional Assay, Western Blot, Expressing, Transfection, Molecular Weight, Flow Cytometry, Positive Control, Concentration Assay, SDS Page, Staining, Purification, Binding Assay, Labeling

    Binding analysis of recombinant TTC and TTC-FITC on isolated PBMCs. Surface staining of CD27 + memory B cells ( a ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( c ) using recombinant TTC (10 nM) and anti-His5 Alexa Fluor 488 antibody (1:100). Surface staining of CD27 + memory B cells ( b ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( d ) using a FITC-coupled TTC peptide (1:25)

    Journal: BMC Biotechnology

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    doi: 10.1186/s12896-016-0249-x

    Figure Lengend Snippet: Binding analysis of recombinant TTC and TTC-FITC on isolated PBMCs. Surface staining of CD27 + memory B cells ( a ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( c ) using recombinant TTC (10 nM) and anti-His5 Alexa Fluor 488 antibody (1:100). Surface staining of CD27 + memory B cells ( b ) and intracellular staining of CD27 ++ CD38 ++ plasma cells ( d ) using a FITC-coupled TTC peptide (1:25)

    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ].

    Techniques: Binding Assay, Recombinant, Isolation, Staining

    Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein

    Journal: BMC Biotechnology

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    doi: 10.1186/s12896-016-0249-x

    Figure Lengend Snippet: Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein

    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ].

    Techniques: Labeling, SDS Page, Fluorescence, In Vivo Imaging, Staining, SDS-Gel, Marker

    Dose-dependent binding analysis of the recombinant fusion protein TTC-ETA’ on hybridoma cells. Various concentrations (1–400 nM) of TTC-ETA’ were used to determine a dose-dependent binding activity on TTC-reactive hybridoma cell line 5E4 ( a ) and to exclude specific binding to the control hybridoma cell line 8.18-C5 ( b ). The detection of bound protein was carried out by flow cytometry using a Penta-His Alexa Fluor 488 Conjugate antibody. Measurements were performed in triplicates (n = 3); error bars indicate SD. The recombinant TTC-ETA’ exhibits a dosedependent binding on the target hybridoma cell line 5E4, whereas no binding could be determined on the control hybridoma cell line 8.18-C5

    Journal: BMC Biotechnology

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    doi: 10.1186/s12896-016-0249-x

    Figure Lengend Snippet: Dose-dependent binding analysis of the recombinant fusion protein TTC-ETA’ on hybridoma cells. Various concentrations (1–400 nM) of TTC-ETA’ were used to determine a dose-dependent binding activity on TTC-reactive hybridoma cell line 5E4 ( a ) and to exclude specific binding to the control hybridoma cell line 8.18-C5 ( b ). The detection of bound protein was carried out by flow cytometry using a Penta-His Alexa Fluor 488 Conjugate antibody. Measurements were performed in triplicates (n = 3); error bars indicate SD. The recombinant TTC-ETA’ exhibits a dosedependent binding on the target hybridoma cell line 5E4, whereas no binding could be determined on the control hybridoma cell line 8.18-C5

    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ].

    Techniques: Binding Assay, Recombinant, Activity Assay, Flow Cytometry, Cytometry

    Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )

    Journal: BMC Biotechnology

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    doi: 10.1186/s12896-016-0249-x

    Figure Lengend Snippet: Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )

    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ].

    Techniques: Binding Assay, Recombinant, Staining, Fluorescence

    Mlph’s phosphorylation state does not interfere substantially with actin binding. ( A ) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left ) and the phosphorylated (Phos; Right ) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. ( B ) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)

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

    Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

    doi: 10.1073/pnas.1619473114

    Figure Lengend Snippet: Mlph’s phosphorylation state does not interfere substantially with actin binding. ( A ) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left ) and the phosphorylated (Phos; Right ) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. ( B ) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)

    Article Snippet: Before the elution of the protein from the Ni-NTA beads, the wash buffer was supplemented with 20 µM SNAP-tag substrate SNAP-Surface Alexa Fluor 647 or SNAP-Surface Alexa Fluor 488 (New England Biolabs) and was added to the Ni-NTA beads.

    Techniques: Binding Assay, Labeling, Incubation, Fluorescence