Recombinant protein (200 μM) was incubated with 5 mM dithiothreitol (DTT) for 1 h at room temperature. DTT was removed via two consecutive passes through a PD-10 Sephadex G-25 M buffer exchange column (GE Healthcare) according to the manufacturer’s instructions into labeling buffer (50 mM HEPES [pH 7.1], 200 mM KCl, 5% glycerol, 120 μM TCEP). Flowthrough was analyzed for protein content at 280 nm. Reduced protein (50 μM) was incubated with 100 μM ATTO 488-maleimide (ATTO-TEC) overnight at 4°C, shielded from light, and subjected to gentle shaking. The reaction was stopped by the addition of 6 mM β-mercaptoethanol, and mixtures were applied to a 1-ml HisTrap HP Ni2+ column (GE Healthcare) before elution using a gradient of buffer B (50 mM Tris [pH 7.5], 200 mM NaCl, 5% glycerol, 500 mM imidazole) and subsequent dialysis to remove the imidazole. Labeling efficiency was calculated in accordance with the fluorescent dye manufacturer’s guidelines.
Pd 10 sephadex g 25 m buffer exchange column
Manufactured by GE Healthcare
The PD-10 Sephadex G-25 M buffer exchange column is a laboratory instrument used for the separation and purification of molecules based on their size. It is designed to efficiently exchange buffers or remove small molecules from larger molecules, such as proteins, peptides, or nucleic acids. The column contains Sephadex G-25 M resin, which allows for effective size-exclusion chromatography.
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2 protocols using pd 10 sephadex g 25 m buffer exchange column
1
Protein Labeling with ATTO 488
2
Recombinant Protein Labeling with ATTO 488
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200 µM recombinant protein was incubated with 5 mM dithiothreitol (DTT) for 1 h at room temperature.
DTT was removed via two consecutive passes through a PD-10 Sephadex G-25 M buffer exchange column (GE Healthcare) as per the manufacturer's instructions into labelling buffer (50 mM HEPES pH 7.1, 200 mM KCl, 5% glycerol, 120 µM TCEP). Flow-through was analysed for protein content at 280 nm. 50 µM of reduced protein was incubated with 100 µM ATTO 488-maleimide (ATTO-TEC) overnight at 4°C, shielded from light and subject to gentle shaking. The reaction was stopped by addition of 6 mM β-mercaptoethanol and mixtures were applied to a 1 ml HisTrap HP Ni 2+ column (GE Healthcare) before elution using a gradient of Buffer B (50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 500 mM imidazole) and subsequent dialysis to remove imidazole. Labelling efficiency was calculated in accordance with the fluorescent dye manufacturer's guidelines.
DTT was removed via two consecutive passes through a PD-10 Sephadex G-25 M buffer exchange column (GE Healthcare) as per the manufacturer's instructions into labelling buffer (50 mM HEPES pH 7.1, 200 mM KCl, 5% glycerol, 120 µM TCEP). Flow-through was analysed for protein content at 280 nm. 50 µM of reduced protein was incubated with 100 µM ATTO 488-maleimide (ATTO-TEC) overnight at 4°C, shielded from light and subject to gentle shaking. The reaction was stopped by addition of 6 mM β-mercaptoethanol and mixtures were applied to a 1 ml HisTrap HP Ni 2+ column (GE Healthcare) before elution using a gradient of Buffer B (50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 500 mM imidazole) and subsequent dialysis to remove imidazole. Labelling efficiency was calculated in accordance with the fluorescent dye manufacturer's guidelines.
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