snap surface alexa fluor  (New England Biolabs)

Journal: Bio-protocol

Article Title: Reconstitution of Active Plant H + -ATPase AHA2 in Giant Unilamellar Vesicles

doi: 10.21769/BioProtoc.5675

Figure Lengend Snippet: Preformed LUVs are destabilized by detergent, followed by the addition of detergent-solubilized, Alexa647-labeled SNAP-AHA2. Free Alexa647 dye and most of the detergent are subsequently removed by size-exclusion chromatography using Sephadex G-50. Residual detergent is removed by adsorption onto bio-beads, yielding purified AHA2-containing proteoliposomes.

Article Snippet: SNAP-Surface ® Alexa Fluor ® 647 (New England Biolabs, catalog number: S9136S) dissolved in DMSO and stored at -20 °C 25.

Techniques: Labeling, Size-exclusion Chromatography, Adsorption, Purification

Journal: Bio-protocol

Article Title: Reconstitution of Active Plant H + -ATPase AHA2 in Giant Unilamellar Vesicles

doi: 10.21769/BioProtoc.5675

Figure Lengend Snippet: (A) Proteo-GUVs were imaged in the pyranine and Alexa647 channels. (B) Each proteo-GUV diameter was measured using ImageJ and plotted. (C) Pyranine fluorescence intensity was quantified for individual proteo-GUVs by placing a region of interest (ROI; yellow dashed circle) within the GUV lumen and measuring the integrated density per pixel using ImageJ. Proteo-GUVs were analyzed over time before and after ATP addition. Scale bar, 20 μm.

Article Snippet: SNAP-Surface ® Alexa Fluor ® 647 (New England Biolabs, catalog number: S9136S) dissolved in DMSO and stored at -20 °C 25.

Techniques: Fluorescence

Journal: Bio-protocol

Article Title: Reconstitution of Active Plant H + -ATPase AHA2 in Giant Unilamellar Vesicles

doi: 10.21769/BioProtoc.5675

Figure Lengend Snippet: Representative Alexa647 fluorescence images and Coomassie Brilliant Blue–stained SDS–PAGE, acquired using a GelDoc system (Bio-Rad), of samples collected at successive stages of the workflow: before labeling (SNAP–AHA2), after the labeling reaction (Alexa647-labeled SNAP–AHA2), before and after size-exclusion chromatography (before and after Sephadex G-50), and after reconstitution into proteoliposomes (proteoliposomes). Comparison of SNAP–AHA2 and Alexa647-labeled SNAP–AHA2 confirms successful labeling of the proton pump, with bands corresponding to the expected molecular weight of SNAP-AHA2 (119 kDa). The presence of bands of identical molecular weight in all samples demonstrates that the fluorophore remains covalently attached throughout purification and reconstitution and confirms the incorporation of Alexa647-labeled SNAP–AHA2 into proteoliposomes. Free Alexa647 dye was efficiently removed by Sephadex G-50 chromatography.

Article Snippet: SNAP-Surface ® Alexa Fluor ® 647 (New England Biolabs, catalog number: S9136S) dissolved in DMSO and stored at -20 °C 25.

Techniques: Fluorescence, Staining, SDS Page, Labeling, Size-exclusion Chromatography, Comparison, Molecular Weight, Purification, Chromatography

Journal: bioRxiv

Article Title: Loss of phosphorylation sites required for β-arrestin recruitment in the calcium-sensing receptor cytoplasmic region may contribute to hypocalcemia

doi: 10.64898/2026.04.21.719673

Figure Lengend Snippet: ( A ) Cartoon showing the CaSR cytoplasmic region for the WT and 895X truncation plasmid. The Thr888 residue that has previously been shown to have a critical role in CaSR signalling is highlighted and was retained in the truncation plasmid. ( B ) Confocal images of the CaSR-WT and CaSR-895X plasmids in HEK-βarr1/2 KO cells. ( C ) Assessment of cell surface expression measured by labelling surface receptor with a CaSR antibody and a secondary Alexa Fluor 647 antibody. Fluorescence was normalized to cells expressing CaSR-WT. ( D ) Ca 2+ i responses measured by fluo-4 with ( E ) EC 50 in cells transfected with CaSR WT or 895X. ( F ) βarrestin-1 (N=9) and ( G ) βarrestin-2 (N=9) recruitment to CaSR in cells expressing CaSR WT and 895X plasmids. AUC was measured to derive concentration-response curves. Statistical analyses were performed by two-way ANOVA with Sidak’s multiple-comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: On the day of the experiment SNAP-Surface Alexa Fluor 647 (NEB) was diluted 1:1000 in FluoroBrite complete media and applied to cells for 20-minutes, before washing and imaging.

Techniques: Plasmid Preparation, Residue, Expressing, Fluorescence, Transfection, Concentration Assay

Journal: bioRxiv

Article Title: Loss of phosphorylation sites required for β-arrestin recruitment in the calcium-sensing receptor cytoplasmic region may contribute to hypocalcemia

doi: 10.64898/2026.04.21.719673

Figure Lengend Snippet: ( A ) SIM of HA-SNAP-CaSR (labelled with the HA antibody and an Alexa Fluor 647 secondary antibody) and Venus-Kras in AdHEK cells. Scale, 5μm. ( B ) Western blot analyses to detect CaSR in cells transfected with mock (pcDNA3.1), FLAG-CaSR-WT (FLAG) and HA-SNAP-CaSR-WT (CaSR) plasmids showing similar expression levels. Calnexin was used as a loading control. The blot shows lysates from three independent transfections. ( C ) Normalized Ca 2+ i signaling measured by Fluo-4 assays. N=5 biological replicates. The maximal response for each drug concentration was derived and normalized to baseline responses of the CaSR-WT transfected wells to derive the concentration-response curve. ( D ) IP-1 responses in cells transfected with HA-SNAP-CaSR and FLAG-CaSR. N=3 biological replicates. ( E ) SIM images of HA-SNAP-CaSR exposed to 5mM Ca 2+ e and the HA antibody for 0 or 30 minutes.

Article Snippet: On the day of the experiment SNAP-Surface Alexa Fluor 647 (NEB) was diluted 1:1000 in FluoroBrite complete media and applied to cells for 20-minutes, before washing and imaging.

Techniques: Western Blot, Transfection, Expressing, Control, Concentration Assay, Derivative Assay

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Biochemical and live-cell imaging characterization of the effects of insulin on INSR. (A) Internalized to total INSR ratio was quantified using surface biotinylation in undifferentiated C2C12 myoblasts. Cells were incubated in PBS (no insulin) or serum-free DMEM containing 0, 0.2, 2, or 20 nM insulin for 15 minutes. The ratios are normalized to the 0 nM group of each gel ( P > .05 when not specified). (B) Western blot showing SNAP-tagged INSR expressed from lentiviral vector in comparison to wild-type INSR in C2C12 myoblasts. (C, D) Representative images of INSR-A-SNAP-labeled using an Alexa Fluor 488 cell nonpermeable dye in 0, 0.2, or 20 nM insulin conditions in live undifferentiated C2C12 myoblasts from (C) 0 to 15 minutes or (D) after 20 minutes using a spinning disk confocal microscope. Time-lapse images of INSR vesicle interactions starting from the selected subregions (white squares) of snapshots are shown in the insets ( n = 3 cells).

Article Snippet: Cell nonpermeable SNAP-Surface Alexa Fluor 488 substrate (Cat. #S9129S, New England Biolabs) was dissolved and diluted to 5 μM concentration in serum-free medium containing 0.5% bovine serum albumin according to the manufacturer's instruction.

Techniques: Live Cell Imaging, Incubation, Western Blot, Plasmid Preparation, Comparison, Labeling, Microscopy