Affinity chromatography medium for separating and purifying GABA receptor and preparation method thereof
A technology for separation, purification and chromatography medium, which is applied in the biological field to achieve the effect of stable and reliable purification method, great application prospect and low price.
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Embodiment 1
[0028] Example 1 Preparation method of fipronil affinity ligand
[0029] Synthesis of product A: Add 4.40 g (10 mmol) fipronil and 30 mL CH 2 Cl 2 , add 2 g of 4-dimethylaminopyridine (DMAP) and 4 mL of triethylamine under ice bath and nitrogen protection, stir for 30 min, add 4 mL of bromoacetyl bromide (about 46 mmol) dropwise, and react at room temperature for 8 h. The reaction solution was poured into 40 mL of ice water and washed with CH 2 Cl 2 (20 mL × 3) extraction, combined organic phase, anhydrous MgSO 4 Dry overnight, precipitate under reduced pressure, and separate by silica gel chromatography [V 石油醚 :V 乙酸乙酯 =8:1] to obtain a yellow solid A with a yield of 70%, m.p. 167-169°C.
[0030] Synthesis of product B: Add 2.32g (0.004mol) of product A, 7.4g (0.04mol) of potassium phthalimide, 0.8g of tetrabutylammonium bromide (TBAB) and 40mL of DMF into a 100mL three-necked flask , Reaction at 80°C for 6h. The reaction solution was poured into 40 mL of ice water and...
Embodiment 2
[0032] Example 2 Preparation method of fipronil affinity ligand
[0033] Synthesis of product A: Add 4.40 g (10 mmol) fipronil and 30 mL CH 2 Cl 2 , add 2 g of 4-dimethylaminopyridine (DMAP) and 4 mL of triethylamine under ice bath and nitrogen protection, stir for 30 min, add 2.6 mL of bromoacetyl bromide (about 30 mmol) dropwise, and react at room temperature for 12 h. The reaction solution was poured into 40 mL of ice water and washed with CH 2 Cl 2 (20 mL × 3) extraction, combined organic phase, anhydrous MgSO 4 Dry overnight, precipitate under reduced pressure, and separate by silica gel chromatography [V 石油醚 :V 乙酸乙酯 =8:1] to obtain a yellow solid A with a yield of 70%, m.p. 167-169°C.
[0034] Synthesis of product B: Add 2.32g (0.004mol) of product A, 3.7g (0.02mol) of potassium phthalimide, 0.8g of tetrabutylammonium bromide (TBAB) and 40mL of DMF into a 100mL three-necked flask , reacted at 80°C for 10 h. The reaction solution was poured into 40 mL of ice water...
Embodiment 3
[0036] Example 3 Preparation of affinity chromatography medium
[0037]Take 5 g of epoxy-activated Sepharose CL-6B gel, washed and drained, and add it to a 50 mL round bottom flask. Add 500 mg of fipronil affinity ligand, 20 mL of 0.1 mol / L sodium carbonate buffer (pH 9.0) and 5 mL of DMSO into the reaction flask, and shake in a water bath at 37 °C for 12 h (attached figure 1 ). Transfer the material liquid to a sand core funnel to drain, wash with deionized water, add 1 mol / L ethanolamine and shake at 37°C for 4 h; Sodium buffer solution, deionized water and 0.1 M pH 8.0 NaAc containing 0.5 M NaCl boric acid-sodium tetraborate buffer solution for washing and washing, and drying after washing to obtain the fipronil ligand affinity chromatography medium of the present invention. The prepared gel was stored in 20% ethanol at 4°C for future use.
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