Bioconjugation of fluorophore (20–150 equiv.) and protein (1 equiv.) was performed in a copper-free environment at room temperature in the dark. The reaction proceeded in 25–100 µL His buffer for up to 4 hours. Protein mutants with the AzPhe were treated with the fluorophore BCN-POE3-NH-DY649P1, and mutants with the NorLys2 were treated with 6-methyl-tetrazine-ATTO-647N. Wild type ACP-GFP served as negative control, treated with the same fluorophores under the same conditions. As reference, 1 (link) equiv. wild type ACP was phosphopantetheinylated with 5 equiv. of the fluorescent substrate CoA 647 (NEB), catalysed by 0.5 equiv. 4′-phosphopantetheinyl transferase Sfp from B. subtilis. The phosphopantetheinylation was performed in presence of 10 mM MgCl2 for 30–45 min at 37 °C in the dark. Subsequent to bioconjugation, an analytical SDS-PAGE was performed and fluorescent protein bands were detected at the Fusion SL Fluorescence Imaging System (Vilber Lourmat) with excitation in the near infrared range and using emission filter F-695 Y5. The FusionCapt Advance Solo 4 16.08a software was used to quantify the fluorescence of the protein bands on the polyacrylamide gel.
Coa 647
Manufactured by New England Biolabs
CoA 647 is a fluorescent dye that can be used for labeling proteins, nucleic acids, and other biomolecules. It has an absorption maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the far-red/near-infrared region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Snap surface alexa fluor 647, by New England Biolabs (2 mentions)
Pd minitrap g 25, by GE Healthcare (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fusion sl fluorescence imaging system, by Vilber (1 mentions)
Baclofen, by Abcam (1 mentions)
Cgp54626, by Bio-Techne (1 mentions)
Snap lumi4 tb, by PerkinElmer (1 mentions)
Snap green, by PerkinElmer (1 mentions)
3 appa, by Abcam (1 mentions)
Cgp54626 red, by PerkinElmer (1 mentions)
Snap red, by PerkinElmer (1 mentions)
Tag lite buffer, by PerkinElmer (1 mentions)
Skf 97541, by Bio-Techne (1 mentions)
Sfp synthase, by New England Biolabs (1 mentions)
4 protocols using coa 647
1
Fluorescent Protein Bioconjugation Protocol
2
Fluorescent Labeling of PLCγ1 Proteins
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Full-length PLCγ1 was labeled with SNAP-Surface Alexa Fluor 647 (S9136S; NEB). Briefly, 10 µM PLCγ1 was mixed with 20 µM SNAP-Ax647 in 500 µl reaction buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, and 10% glycerol). Following incubation in the dark for 2 h at 37°C, the labeled protein was separated from the solution by size-exclusion chromatography using PD MiniTrap G25 (GE Healthcare).
PLCγ1 nSH2-cSH2-SH3 and truncations were labeled with CoA 647 (S9350; NEB) via an N-terminal ybbR tag. 10 µM ybbR-fusion proteins were mixed with 20 µM CoA-647 and 2 µM GST-Sfp in 200 µl reaction buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, 10 mM MgCl2, and 10% glycerol). After incubation for 2 h at room temperature, 20 µl Glutathione Sepharose 4B was added to deplete GST-Sfp. The supernatant was further applied to size-exclusion chromatography using PD MiniTrap G25 and stored in 50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM TCEP, and 10% glycerol.
PLCγ1 nSH2-cSH2-SH3 and truncations were labeled with CoA 647 (S9350; NEB) via an N-terminal ybbR tag. 10 µM ybbR-fusion proteins were mixed with 20 µM CoA-647 and 2 µM GST-Sfp in 200 µl reaction buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, 10 mM MgCl2, and 10% glycerol). After incubation for 2 h at room temperature, 20 µl Glutathione Sepharose 4B was added to deplete GST-Sfp. The supernatant was further applied to size-exclusion chromatography using PD MiniTrap G25 and stored in 50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM TCEP, and 10% glycerol.
3
Labeling of PLC1 and Truncations
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The full-length PLC1 was labeled with SNAP-Surface Alexa Fluor 647 (NEB, S9136S).
Briefly, 10 μM PLC1 was mixed with 20 μM SNAP-Ax647 in 500 μL reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, and 10% glycerol).
Following incubation in the dark for 2 hrs at 37 º C , the labeled protein was separated from the solution by size exclusion chromatography using PD MiniTrap G25 (GE Healthcare).
PLC1 nSH2-cSH2-SH3 and truncations were labeled with CoA 647 (NEB, S9350) via an N-terminal ybbR tag. 10 μM ybbR-fusion proteins were mixed with 20 μM CoA-647 and 2 μM GST-Sfp in 200 μL reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, 10 mM MgCl2 and 10% glycerol). After an incubation of 2 hrs at room temperature, 20 μL Glutathione Sepharose 4B was added to deplete GST-Sfp. The supernatant was further applied to size exclusion chromatography using PD MiniTrap G25 and stored in 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP and 10% glycerol.
Briefly, 10 μM PLC1 was mixed with 20 μM SNAP-Ax647 in 500 μL reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, and 10% glycerol).
Following incubation in the dark for 2 hrs at 37 º C , the labeled protein was separated from the solution by size exclusion chromatography using PD MiniTrap G25 (GE Healthcare).
PLC1 nSH2-cSH2-SH3 and truncations were labeled with CoA 647 (NEB, S9350) via an N-terminal ybbR tag. 10 μM ybbR-fusion proteins were mixed with 20 μM CoA-647 and 2 μM GST-Sfp in 200 μL reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 1 mM DTT, 10 mM MgCl2 and 10% glycerol). After an incubation of 2 hrs at room temperature, 20 μL Glutathione Sepharose 4B was added to deplete GST-Sfp. The supernatant was further applied to size exclusion chromatography using PD MiniTrap G25 and stored in 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP and 10% glycerol.
4
GABA receptor binding assay
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Materials and Reagents GABA, CGP54626, and SKF97541 were purchased from Tocris Bioscience. Baclofen, 3-APPA, CGP35348, CGP7930, GS39783, and rac-BHFF were purchased from Abcam. Lipofectamine 2000 and Fluo-4 AM were from Thermo Fischer Scientific. SNAP-Lumi4-Tb, SNAP-Green, SNAP-Red, CoA-Lumi4-Tb, and Tag-Lite buffer can be purchased from Cisbio Bioassays. CoA-Alexa 488, CoA-647, and Sfp synthase were from New England Biolabs. Benzyl guanine (BG) derivative fluorophores BG-Green-LC and BG-Atto465, and CGP54626-Red were a gift from Cisbio Bioassays.
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