High-load agarose chromatography media and preparation method thereof
A chromatographic medium and agarose technology, which is applied in the field of high-load agarose chromatographic medium and its preparation, can solve problems such as preventing proteins from entering the medium, and achieve increased medium load, increased number of sites, and protein loading. The effect of volume increase
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Embodiment 1
[0033] One-time activation: Accurately weigh 10g of Sepharose6FF (6% agarose solution, cross-linking matrix), add it to a three-hole kettle, add 0.01g sodium borohydride, 1g anhydrous sodium sulfate, 2mL30% NaOH solution, 1mL allyl glycidyl ether and 3mL of water, the system was reacted at 50°C for 18h. After the reaction stopped, the medium was repeatedly washed with a large amount of deionized water. Prepare an allyl-activated medium with an allyl density of 150 μmol / g to dry the wet glue.
[0034] Primary bromination: add 20 mL of water, 1.2 g of sodium acetate, and 2 mL of bromine water to 10 g of allyl activation medium, and react for 1 h at room temperature. After the reaction, the medium was washed with plenty of water. Obtain a partially brominated agarose medium with an allyl density of 105 μmol / g and drain the wet gel.
[0035] Grafting dextran: Take 10g of partially brominated agarose medium, add 15mL of 0.6g / ml dextran solution (dextran T40, molecular weight 40k...
Embodiment 2
[0042] Primary activation: Accurately weigh 10 g of cross-linked white spheres prepared with a concentration of 2% agarose solution, and the rest are the same as in Example 1. Prepare an allyl-activated medium with an allyl density of 10 μmol / g to dry the wet glue.
[0043] Primary bromination: Add 10 g of allyl-activated medium to 20 mL of water, 1.2 g of sodium acetate, and 1 mL of bromine water, and react for 1 h at room temperature. After the reaction, the medium was washed with plenty of water. Obtain a partially brominated agarose medium with an allyl density of 1 μmol / g and drain the wet gel.
[0044] Graft dextran: Take 10g of partially brominated agarose medium, add 15mL of 0.6g / ml dextran solution (dextran T2, molecular weight 2kDa), 7mL of water, 10mL of 1M NaOH solution (3.6mg of sodium borohydride), at 30 The reaction was carried out at ℃ for 18 hours, and the medium was washed with a large amount of deionized water after the reaction. Prepare a dextran-grafted...
Embodiment 3
[0051] One-time activation: Accurately weigh 10 g of cross-linked white balls prepared by 15% agarose solution concentration, use allyl bromide as the activator, add 0.01 g of sodium borohydride, 1 g of anhydrous sodium sulfate, 2 mL of 30% NaOH solution, and 3 mL of alkene Propyl glycidyl ether and 3mL water were reacted at 50°C for 12h. An allyl-activated medium was prepared with an allyl density of 2000 μmol / g to dry the wet glue.
[0052] Primary bromination: add 20 mL of water to 10 g of allyl activation medium, 1.2 g of sodium acetate, and 3 mL of bromine water, and react for 1 h at room temperature. After the reaction, the medium was washed with plenty of water. Obtain a partially brominated agarose medium with an allyl density of 1900 μmol / g and drain the wet gel.
[0053] Grafting dextran: Take 10g of partially brominated agarose medium, add 15mL of 0.6g / ml dextran solution (dextran T150, molecular weight 150kDa), 7mL of water, 20mL of 1M NaOH solution (3.6mg sodium...
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