Gibson assembly (56 (link)) was used to clone in-frame a C-terminal SNAP-tag on PfK5ΔL6-MD (PfK5ΔL6-MD-SNAP) for use in total internal reflection microscopy (TIRFM) experiments. Expression and purification of PfK5ΔL6-MD-SNAP were performed as for PfK5ΔL6-MD, except the anion exchange chromatography step was altered as follows: concentrated and desalted sample was added to a 1 ml HiTrap Q HP column, washed with 10 CV IEX W buffer, and eluted with a 20 CV gradient elution to 500 mM NaCl. Eluted fractions containing PfK5ΔL6-MD-SNAP were pooled. PfK5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to PfK5ΔL6-MD-SNAP. Free SNAP-ligand was removed by two repeats of buffer exchange in T50K20 buffer (0.5 ml Zeba 7K MWCO spin columns).
Snap surface alex fluor 647
Manufactured by New England Biolabs
SNAP-Surface Alex Fluor 647 is a synthetic fluorescent dye that can be covalently attached to SNAP-tag fusion proteins. It is designed for labeling and detection applications in biological research.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using snap surface alex fluor 647
1
PfK5ΔL6-MD-SNAP: TIRFM-Ready Protein
2
Cloning and Labeling PfK5ΔL6-MD-SNAP
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Gibson assembly 57 was used to clone in-frame a C-terminal SNAP-tag on PfK5ΔL6-MD (PfK5ΔL6-MD-SNAP) for use in total internal reflection microscopy (TIRFM) experiments. Expression and purification of PfK5ΔL6-MD-SNAP was performed as for PfK5ΔL6-MD, except the anion exchange chromatography step was altered as follows: concentrated and desalted sample was added to a 1 mL HiTrap Q HP column, washed with 10 CV IEX W buffer, and eluted with a 20 CV gradient elution to 500 mM NaCl. Eluted fractions containing PfK5ΔL6-MD-SNAP were pooled. PfK5ΔL6-MD-SNAP was biotinylated or fluorescently labelled by overnight incubation at 4 o C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 molar excess of these labels to PfK5ΔL6-MD-SNAP. Free SNAP-ligand was removed by 2 repeats of buffer exchange in T50K20 buffer (0.5 mL Zeba 7K MWCO spin columns).
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