snap surface alexa fluor 546  (New England Biolabs)

Journal: Analytical biochemistry

Article Title: An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

doi: 10.1016/j.ab.2016.05.026

Figure Lengend Snippet: Monitoring 26S proteasome activity by fluorescence anisotropy. Degradation of the polyubiquitinated Alexa Fluor 546-labeled substrate by purified yeast proteasome as monitored by fluorescence anisotropy measurements. The degradation reaction was monitored

Article Snippet: We purified the UbL substrates from E. coli , labeled with SNAP-surface Alexa Fluor 546, and conducted the fluorescence anisotropy assay with purified yeast proteasome as described above.

Techniques: Activity Assay, Fluorescence, Labeling, Purification

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: Transient dimer formation of SNAP-D 2L receptors. ( a ) 48 sequential frames of two Alexa546-labeled SNAP-D 2L receptors showing transient dimer formation (frame rate of 19.32 fps) (also shown in Supplementary Movie S3 ). ( b ) Intensity profile (blue) of the marked fluorescent SNAP-D 2L receptor shown in ( a) , compared to background intensity (grey). ( c ) A histogram of the lifetimes of 120 SNAP-D 2L receptor dimers taken from trajectories similar to those in ( a) and collected in 0.5 s bins. The solid line represents a one-phase exponential fit for a mean lifetime of 0.50 s (95% confidence interval: 0.44–0.60). ( d ) Effect of the size of SNAP-D 2L receptors on their lateral diffusion. The diffusion coefficients (D lat ) of the analyzed receptor particles ( n ) are shown (monomers −0.104 ± 0.052 μm 2 s −1 , n = 412 from 3 cells and dimers −0.075 ± 0.027 μm 2 s −1 , n = 373 from 3 cells. Data represent mean ± s.d. The difference, determined by an unpaired t-test (**** p -value

Article Snippet: SNAP-tag labeling Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface® Alexa Fluor® 546; New England Biolabs).

Techniques: Labeling, Diffusion-based Assay

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: Visualization, diffusion and dimerization of Alexa546-labeled SNAP-D 2L receptors and on membrane protrusions using HF-treated slides. ( a ) Representative images of a single CHO cell stably expressing the SNAP-D 2L receptor and seeded on HF-treated glass slides, labeled with Alexa546-BG and visualized by TIRF-M. The insert corresponds to higher magnification image of the area in the small white box. ( b ) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in a. The plot is linear (r 2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk. ( c ) The distribution of the diffusion coefficients of the analyzed receptor particles ( n ) are shown ( n = 4409, 5 cells - HF-treated slide; n = 4409, 8 cells - non HF-treated slide) and revealed no evidence for anomalous diffusive behavior as a result of HF-treatment. ( d ) Representative intensity distribution of fluorescent spots identified over the first 10-frame time window of TIRF-illumination. Data were fitted with a mixed Gaussian model (sum of two Gaussian functions). ( e ) Representative TIRF-M images of CHO cells stably transfected with SNAP-D 2L receptor, incubated in iso-osmotic (300 mOsm) (left) and hypo-osmotic PBS (108 mOsm) (right) for 2 h and labeled with Alexa546-BG. ( f,g ) Imaging of a region of membrane protrusions of CHO cells stably transfected with the SNAP-D 2L receptor, incubated in hypo-osmotic (108 mOsm) PBS for 2 h and labeled with Alexa546-BG in epi-illumination ( f ) and TIRF-illumination ( g ). ( h ) Representative images of one membrane protrusion of a CHO cell stably expressing the labeled SNAP-D 2L receptor and visualized by TIRF-M. ( f ) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in ( h ). The plot is linear (r 2 = 0.99 – linear fit (blue)), over a 2,5-s time scale and the calculation of the average diffusion coefficient D lat of 0.077 ± 0.007 μm 2 s −1 (mean ± s.d., 16 regions of membrane protrusions of 8 cells) revealed no evidence for anomalous diffusive behavior in the membrane protrusions. Scale bars, 10 μm.

Article Snippet: SNAP-tag labeling Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface® Alexa Fluor® 546; New England Biolabs).

Techniques: Diffusion-based Assay, Labeling, Stable Transfection, Expressing, Transfection, Incubation, Imaging

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: Influence of monovalent (1a,b), bivalent (2a,b) and bivalent control (3a,b) dopamine D 2 receptor antagonists on receptor dimerization. ( a ) Chemical structures of monovalent ligands ( 1a–c ), bivalent ligands ( 2a–c ), and control ligands ( 3a,b ). ( b ) Monomer/dimer ratios calculated from fitted fluorescence intensity distributions of Alexa546-labeled SNAP-D 2L receptors incubated with monovalent ( 1a,b ), bivalent ( 2a,b ), control ( 3a,b ) ligands using a mixed Gaussian model ( Supplementary Table S2 , Supplementary Fig. S6a ). Data represent mean ± s.d. of n analysed cells ( n = 16 for 1a , 8 for 1b , 8 for 2a , 16 for 2b , 6 for 3a and 8 for 3b . ( c ) Average diffusion coefficients (D lat ) of the corresponding ligand-SNAP-D 2L receptor complexes of the same analyzed cells in ( b ). Data in ( b,c ) represent mean ± s.d., Statistical analysis was performed by an unpaired t -test (** p -value

Article Snippet: SNAP-tag labeling Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface® Alexa Fluor® 546; New England Biolabs).

Techniques: Fluorescence, Labeling, Incubation, Diffusion-based Assay

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: Visualization, tracking and analysis of the dimerization of single SNAP-tagged D 2L receptors using SNAP-CD86 and SNAP-CD28 as monomeric and dimeric reference proteins. ( a,g,j ) Schematic representation of the SNAP-tagged constructs. ( b,h,k ) Representative images of single CHO cells, stably transfected with the corresponding labeled protein and visualized by TIRF-M. Scale bar, 10 μm. The first 100 frames of the cell in b are shown in Supplementary Movie S1 . Inserts correspond to higher magnification images of the areas in the white boxes. ( c,i,l ) Representative intensity distributions of fluorescent spots identified over the first 10-frame time window of TIRF illumination of CHO cells, stably transfected with the corresponding construct and labeled with Alexa546-BG. Number of identified particles, n = 5770 ( c ), 6252 ( f ) and 6458 ( i ). Data were fitted with a mixed Gaussian model. A mixed Gaussian fit after partial photobleaching (dotted line) was used to estimate the intensity of a single fluorescent molecules in each image sequence. ( d ) Individual trajectories of moving SNAP-D 2L receptors were identified from the entire recording of the cell shown in ( b) . The insert shows a higher magnification that illustrates the random nature of the diffusive process. ( e ) Representative plot of the average mean square displacement (MSD) (mean ± s.d.) versus the time interval (δt) for the trajectories shown in ( a ). The plot is linear (r 2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk, and it shows no evidence for anomalous diffusive behavior. ( f ) Distribution of the diffusion coefficients of the receptor particles tracked in ( d ).

Article Snippet: SNAP-tag labeling Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface® Alexa Fluor® 546; New England Biolabs).

Techniques: Construct, Stable Transfection, Transfection, Labeling, Sequencing, Diffusion-based Assay

Journal: Biophysical Journal

Article Title: Effect of Phosphorylation on EGFR Dimer Stability Probed by Single-Molecule Dynamics and FRET/FLIM

doi: 10.1016/j.bpj.2015.01.005

Figure Lengend Snippet: Kinetic analysis of EGFR homodimer dissociation. ( a ) Distribution of EGFR homodimer lifetimes after EGF stimulation for all accumulated data acquired in five individual experiments employing various TKIs (gefitinib, lapatinib, and AZD 8931). The distribution of more than 2000 dimerization events was fitted to a monoexponential where k app EGF = 1.19 ± 0.05 s −1 . ( b ) Two-color fluorescence emission corresponding to a pair of colocalized molecules labeled with Alexa Fluor 546 ( green ) and Atto 647 ( red ), respectively. ( c ) Changes with time of the horizontal and vertical positions of the dimerization partners. The two molecules temporally coexist between ∼0 and 12 s from the beginning of the measurement. Dimer formation implies simultaneous detection of both molecules at the same time (temporal colocalization) and same location (spatial colocalization). The horizontal and vertical feature positions report on the molecules’ spatial colocalization. ( d ) The separation distance between the two-color monomers in proximity to each other decreases below the threshold and marks the start of the dimerization event. For the duration of the dimerization event, the separation distance remains under the threshold, followed by a stepwise increase above the threshold upon dimer dissociation. This allows us to extract the apparent duration of an individual dimerization event, τ app . The photobleaching corrected colocalization duration provides the τ on for the EGFR homodimer and the corresponding dissociation rate k off . To see this figure in color, go online.

Article Snippet: The antibody was fluorescently labeled with either Snap-Alexa Fluor 546 or Snap-Alexa Fluor 647 (New England Biolabs, UK) with a labeling stoichiometry of 0.3 and 0.9 dye molecules/protein, respectively.

Techniques: Fluorescence, Labeling

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. ( A ) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. ( B ) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p

Article Snippet: SNAP surface Alexa Fluor-546 and SNAP Surface Block were from New England Biolabs, UK.

Techniques: Expressing, Mutagenesis, Incubation, Microscopy, Fluorescence, Western Blot

Journal: Scientific Reports

Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

doi: 10.1038/s41598-019-53176-4

Figure Lengend Snippet: Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.

Article Snippet: SNAP surface Alexa Fluor-546 and SNAP Surface Block were from New England Biolabs, UK.

Techniques: Expressing, Incubation, Immunostaining, Staining, Microscopy, Binding Assay