BG-PEG-NH 2 (SNAP-PEG-NH 2 ) is an amino-terminal fluorescent probe building block and a precursor for site-specific labeling of SNAP-tag fusion proteins. BG-PEG-NH 2 undergoes one-step coupling with activated carboxyl esters to form customized SNAP-tag substrates, which covalently bind to SNAP tags via a thioether bond formed at the Cys145 site. After coupling with a caged carboxyfluorescein, BG-PEG-NH 2 forms a probe with Ex/Em = 500/524 nm following ultraviolet uncaging. Coupling of BG-PEG-NH 2 with SeTau-647-NHS enables visualization of GPCR signal transduction. BG-PEG-NH 2 is suitable for coupling reactions in aqueous phases or buffers, conjugation of labels, and labeling of intracellular or cell-surface proteins [1] [2] [3].
In Vitro, Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs) 1. Stock Solution Preparation 1.1 Solvent: DMSO; Alternative: RNase-free DMSO. 2. Working Solution Preparation 2.1 Diluent: Complete cell culture medium; Alternative: HBSS (+). 2.2 Working concentration: 50 nM or 5 μM. 2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately. 3. Staining Procedure 3.1 Sample Types: 3.1.1 Adherent mammalian cells expressing SNAP-tag fusion protein[1][3]: 3.1.1.1 No trypsin digestion is required before staining; some cell lines need to be cultured in collagen gel-coated glass-bottom dishes/plates. 3.2 Incubation Conditions: For adherent mammalian cells expressing SNAP-tag fusion protein: Incubate the cells with the working solution at 37°C under 5% CO2 for 15-30 min. 3.3 Washing Steps: For adherent mammalian cells expressing SNAP-tag fusion protein: Wash the cells twice with complete medium before staining; if optional nuclear counterstaining is performed, wash the cells three times with complete medium after counterstaining; alternatively, wash the cells twice with HBSS (+), followed by incubation in a CO2 incubator for 15 min, and then replace with FluoroBrite DMEM medium supplemented with 1% FBS. 3.4 Nuclear Counterstaining (Optional): Incubate the cells with Hoechst 33342 solution at 37°C under 5% CO2 for 3 min. 4. Control Setup 4.1 Use untransfected or mock-transfected cells as negative controls. 4.2 Block the SNAP-tag activity of transfected cells with SNAP-Cell Block as an additional negative control. 5. Detection and Analysis 5.1 Instrument Types: Fluorescence microscope, confocal laser scanning microscope, total internal reflection fluorescence microscope (TIRFM). 5.2 Excitation/Emission Wavelengths 5.2.1 For BG-CMNB-caged carboxyfluorescein (derived from BG-PEG-NH2): Ex 500 nm / Em 524 nm. 5.2.2 For SeTau647-PEG-BG (derived from BG-PEG-NH2): Ex 640 nm / Em 697/58 nm. 5.3 Result Analysis 5.3.1 Detect stable fluorescent signals localized to subcellular regions of the SNAP-tag fusion protein (e.g., nucleus, basolateral membrane, early endosome, cell membrane). 5.3.2 If a nuclear counterstain is used, the nucleus shows blue fluorescence. 5.3.3 Under optimal conditions, the fluorescent signal can be detected for up to 48 h after labeling. 5.3.4 For caged fluorophore derivatives: Green fluorescence is observed after ultraviolet deprotection, and this signal persists through cell division, which can be used for cell lineage tracing. 5.3.5 The fluorescent signal remains detectable after fixation with acidic fixatives (e.g., 10% trichloroacetic acid, methanol). 5.3.6 For labeled SNAP-FPR1: Quantify the fluorescence intensity of FPR1 spots with a threshold of 900 counts to distinguish small oligomers from large oligomers; stimulation with a full agonist increases the proportion of large FPR1 oligomers, and ligand-bound large oligomers show longer colocalization with Gαi3 compared to unliganded or small oligomer states.
References:
[1]. Corrêa IR, et al. Considerations and protocols for the synthesis of custom protein labeling probes. Methods in molecular biology (Clifton, N.J.). 2015;1266:55-79.
[2]. Campos C, et al. Labelling cell structures and tracking cell lineage in zebrafish using SNAP-tag. Developmental dynamics: an official publication of the American Association of Anatomists. 2011 Apr;240(4):820-7.
[3]. Nishiguchi T, et al. Synergetic Roles of Formyl Peptide Receptor 1 Oligomerization in Ligand-Induced Signal Transduction. ACS chemical biology. 2020 Sep 18;15(9):2577-2587.
In Vitro, Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs) 1. Stock Solution Preparation 1.1 Solvent: DMSO; Alternative: RNase-free DMSO. 2. Working Solution Preparation 2.1 Diluent: Complete cell culture medium; Alternative: HBSS (+). 2.2 Working concentration: 50 nM or 5 μM. 2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately. 3. Staining Procedure 3.1 Sample Types: 3.1.1 Adherent mammalian cells expressing SNAP-tag fusion protein[1][3]: 3.1.1.1 No trypsin digestion is required before staining; some cell lines need to be cultured in collagen gel-coated glass-bottom dishes/plates. 3.2 Incubation Conditions: For adherent mammalian cells expressing SNAP-tag fusion protein: Incubate the cells with the working solution at 37°C under 5% CO2 for 15-30 min. 3.3 Washing Steps: For adherent mammalian cells expressing SNAP-tag fusion protein: Wash the cells twice with complete medium before staining; if optional nuclear counterstaining is performed, wash the cells three times with complete medium after counterstaining; alternatively, wash the cells twice with HBSS (+), followed by incubation in a CO2 incubator for 15 min, and then replace with FluoroBrite DMEM medium supplemented with 1% FBS. 3.4 Nuclear Counterstaining (Optional): Incubate the cells with Hoechst 33342 solution at 37°C under 5% CO2 for 3 min. 4. Control Setup 4.1 Use untransfected or mock-transfected cells as negative controls. 4.2 Block the SNAP-tag activity of transfected cells with SNAP-Cell Block as an additional negative control. 5. Detection and Analysis 5.1 Instrument Types: Fluorescence microscope, confocal laser scanning microscope, total internal reflection fluorescence microscope (TIRFM). 5.2 Excitation/Emission Wavelengths 5.2.1 For BG-CMNB-caged carboxyfluorescein (derived from BG-PEG-NH2): Ex 500 nm / Em 524 nm. 5.2.2 For SeTau647-PEG-BG (derived from BG-PEG-NH2): Ex 640 nm / Em 697/58 nm. 5.3 Result Analysis 5.3.1 Detect stable fluorescent signals localized to subcellular regions of the SNAP-tag fusion protein (e.g., nucleus, basolateral membrane, early endosome, cell membrane). 5.3.2 If a nuclear counterstain is used, the nucleus shows blue fluorescence. 5.3.3 Under optimal conditions, the fluorescent signal can be detected for up to 48 h after labeling. 5.3.4 For caged fluorophore derivatives: Green fluorescence is observed after ultraviolet deprotection, and this signal persists through cell division, which can be used for cell lineage tracing. 5.3.5 The fluorescent signal remains detectable after fixation with acidic fixatives (e.g., 10% trichloroacetic acid, methanol). 5.3.6 For labeled SNAP-FPR1: Quantify the fluorescence intensity of FPR1 spots with a threshold of 900 counts to distinguish small oligomers from large oligomers; stimulation with a full agonist increases the proportion of large FPR1 oligomers, and ligand-bound large oligomers show longer colocalization with Gαi3 compared to unliganded or small oligomer states.
References:
[1]. Corrêa IR, et al. Considerations and protocols for the synthesis of custom protein labeling probes. Methods in molecular biology (Clifton, N.J.). 2015;1266:55-79.
[2]. Campos C, et al. Labelling cell structures and tracking cell lineage in zebrafish using SNAP-tag. Developmental dynamics: an official publication of the American Association of Anatomists. 2011 Apr;240(4):820-7.
[3]. Nishiguchi T, et al. Synergetic Roles of Formyl Peptide Receptor 1 Oligomerization in Ligand-Induced Signal Transduction. ACS chemical biology. 2020 Sep 18;15(9):2577-2587.