SNAP-tagged proteins were labeled with SNAP-Surface Alexa Fluor 488, SNAP-Surface Alexa Fluor 546, and SNAP-Surface Alexa Fluor 647 (New England Biolabs). Protein (5 µM) and dye (10 µM) were mixed and allowed to react in 50KMEH5Gd at RT for 2 hr. Labeled proteins were desalted into 50KMEH5Gd buffer and concentrated. Extinction coefficients of fluorophores were calculated from a standard curve and are as follows: Alexa 488 at 495 nm, 95,000 M–1*cm–1; Alexa 546 at 556 nm, 120,000 M–1*cm–1; Alexa 647 at 650 nm, 255,000 M–1*cm–1. Protein labeling efficiency was calculated by dividing protein concentration by dye concentration—for Alexa Fluor 488 the labeling efficiency was estimated at ~100%, for Alexa Fluor 546 the labeling efficiency was estimated at ~60%.
Snap surface alexa fluor 546
Manufactured by New England Biolabs
The SNAP-Surface Alexa Fluor 546 is a fluorescent labeling reagent designed for use with SNAP-tag fusion proteins. It provides a simple and efficient way to label SNAP-tag fusion proteins with the Alexa Fluor 546 fluorescent dye.
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7 protocols using snap surface alexa fluor 546
1
Fluorescent labeling of SNAP-tagged proteins
2
Fluorescent Labeling and Ligand Treatment
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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). Subsequently, cells were washed three times with phenol red-free DMEM/F12 supplemented with 10% FBS, each time followed by 5 min incubation at 37 °C.
For the ligand treatment experiments, the indicated concentration of the corresponding ligand in phenol red-free DMEM/F12 supplemented with 10% FBS were added to stably transfected CHO cells, which were ready for imaging, for 1 h before imaging.
For the ligand treatment experiments, the indicated concentration of the corresponding ligand in phenol red-free DMEM/F12 supplemented with 10% FBS were added to stably transfected CHO cells, which were ready for imaging, for 1 h before imaging.
3
Fluorescent Labeling of Receptor Constructs
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Receptor constructs were expressed in HEK 293 T cells for 24–48 hr prior to labeling with 1 µM SNAP-Surface Alexa Fluor 546 (NEB) in extracellular solution at 37°C for 45 min. For two-color experiments with co-expression of SNAP- and CLIP-tagged receptors, cells were labeled with 1 µM BG-LD655 followed by 2 µM BC-DY547 (NEB). After labeling, cells were washed in extracellular solution to remove excess dye. Next, cells were imaged with a 60x objective on an inverted microscope (Olympus IX73). ImageJ was used to analyze fluorescence intensity of cell clusters. Multiple independent experiments were performed for each construct tested, including at least two separate transfections. Fluorescence intensity values for each construct were normalized to wild-type values for comparison.
4
Purification and Labeling of UbL-SNAP Substrates
5
Dynein Constructs for Single-Molecule Studies
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For the monomeric dynein construct MD380, the C-terminal 380-kD motor domain of cytoplasmic dynein from the cellular slime mold D. discoideum was fused to a His6 tag, a FLAG tag, and a biotin tag in tandem at its N terminus, with an additional SNAP tag inserted at the AAA2 domain (Numata et al., 2011 (link)). For dimeric dynein GST380, GST was further inserted between the biotin tag and motor domain. To create MTBD-removed monomeric construct MD380ΔMTBD, the MTBD coding region A3372-K3495 was replaced by linker sequence TG (Kon et al., 2012 (link); also see Fig. S3 A). These dynein constructs were expressed in D. discoideum and purified by His6 and FLAG tag affinity column chromatography (Kon et al., 2009b (link)). For fluorescent labeling, SNAP-Surface Alexa Fluor 546 (New England BioLabs, Inc.) was covalently attached to the dynein SNAP tag. For the dynein MTBD construct (Fig. S3 A), synthetic DNA was optimized for expression in Escherichia coli. Purification was performed using a His6 tag affinity column and gel filtration chromatography.
6
Characterization of G3BP1-RNA Condensates
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SNAP-tagged G3BP1 was mixed with SNAP-Surface Alexa Fluor 546 (NEB) at a ratio of 20:1 at RT for 5 min. To remove free dye, the protein was buffer exchanged using Protein Desalting Spin Columns (ThermoFisher) into 50 mM Tris/HCl, pH 7.5, 300 mM KCl and 1 mM DTT. SNAP(546)-G3BP1-RNA condensates were formed at a concentration of 5 μM G3BP1(WT) and 25 ng/μl total RNA in 20 mM PIPES, pH 7.0, 85 mM KCl, 1 mM MgCl2 and 1% (w/v) PEG-20K. Thereafter, 150 nM GFP-tagged client proteins were added. Samples were mounted on pegylated glass slides and imaged after 20 min. Client enrichment in G3BP1 droplets (ratio of client mean fluorescence intensity inside of G3BP1 droplets over client mean fluorescence intensity outside of droplets) was quantified using the FIJI software and subsequently plotted using RStudio software package. For Caprin-1 partitioning assays, 2.5 μM mCherry-Caprin-1 were added to preformed GFP-G3BP1(WT)-RNA condensates.
7
Visualizing SMO Endocytosis Dynamics
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For the pulse-chase experiments, dissected wing imaginal discs were incubated for 10 min at 25°C with SNAP-Surface Alexa Fluor 546 (3.3 µM, from NEB), rinsed and incubated for 15 min with SNAP-Surface Block at 13 µM before fixation and immunolabeling. For the study of the endocytosis of SMO, shi ts /Y; apGal4/+; UAS SNAP-smo WT /+ and shi + /Y; apGal4/+; UAS SNAP-smo WT /+ wandering L3 larvae were incubated 30°C for 30 min, before being dissected.
After dissection, the wing imaginal discs were incubated with SNAP-Surface Alexa Fluor 546 for 10 min at 30°C before fixation and immunolabeling. apGal4, gal80 ts /UAS yfp-rab5 CA ; UAS SNAP-smo WT /+ and apGal4, gal80 ts /+; UAS SNAP-smo WT /+ larvae were kept at 18°C (P Zeidler, et al., 2004) for 7-8 days. Vials were then moved to 29°C to induce the GAL4-dependent transcription of the UAS constructs for 24 h.
After dissection, the wing imaginal discs were incubated with SNAP-Surface Alexa Fluor 546 for 10 min at 30°C before fixation and immunolabeling. apGal4, gal80 ts /UAS yfp-rab5 CA ; UAS SNAP-smo WT /+ and apGal4, gal80 ts /+; UAS SNAP-smo WT /+ larvae were kept at 18°C (P Zeidler, et al., 2004) for 7-8 days. Vials were then moved to 29°C to induce the GAL4-dependent transcription of the UAS constructs for 24 h.
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