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Epoxy-activated Crystarose 6FF

classification：Pre-activation chromatography

Epoxy-activated Crystarose is an epoxy-activated agarose gel 6FF for coupling substances with free sulfhydryl, amino and hydroxyl groups.

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Introduction

Epoxy-activated Crystarose is an epoxy-activated agarose gel 6FF for coupling substances with free sulfhydryl, amino and hydroxyl groups. Epoxy-based coupling needs to be coupled under relatively harsh conditions, and higher temperature and higher pH can improve the coupling efficiency. Epoxy substrates are mainly used to couple some small molecules, but also can be used to couple more stable large molecules such as proteins. The epoxy ether bond after coupling is very stable and generally not easy to fall off, which is a very ideal coupling method. The products are packed in 25ml, 50ml and bigger packages, the products are kept in the solution containing DMSO, also in the form of lyophilized powder, the packages are 10g, 100g.

II. Filler properties:

Features	Wide range of use, easy to operate
Scope of application	Substances with amino, sulfhydryl, and hydroxyl groups (polysaccharides, proteins, nucleic acids, antibodies, and small molecules)
Substrate	Cross-linked agarose gel
Functional groups	epoxy
Density of matching base	20-40μmol/ml
Grain size	45-160umμm
Maximum flow rate	450cm/h (15cm column height, DDH2O as mobile phase)
Coupling conditions	pH 8-14 Temperature 4-45 degrees, time 1-16 hours, can be coupled in aqueous or organic phase
Products	DMSO preserved or dry powder (approx. 4 ml/g)
Save	Dry powder 4-8℃ dry storage

Scope of application

Coupling small molecules into specific group chromatography packing, or coupling antigen and antibody into affinity chromatography packing

应用实例

实验名称：偶联牛血清白蛋白（BSA）

实验步骤：

1、取 BSA 100mg 溶解于 10ml 的 pH8.5 碳酸钠缓冲液（0.1M）中；

2、用天平称量需要的活化填料 2g，（1g 干的填料经完全溶胀后约4ml左右）,然后用

100 毫升水分三次抽滤清洗；

3、将填料加入溶解后的 BSA 溶液中混合，在摇床上振荡，25℃偶联 6 小时；

4、然后加 20ml 1M Tris-cl PH8.5 ，再反应 6 个小时封闭剩余的基团。

5、偶联了 BSA 的填料可以装在柱子中，然后用0.5M NaCl 溶液洗 10 个柱床体积，收集装柱子的液体以及清柱子的溶液测定 BSA 的总量。再用PH4.0的醋酸盐缓冲溶液（含0.5M NaCL）和PH8.5的碳酸盐缓冲溶液（含0.5M NaCL）循环冲洗3遍，去离子水冲 5 个柱床体积，20%乙醇冲洗2个柱体积，最后将填料在 20%乙醇溶液中保存。

6、 BSA 的偶联量计算方法：BSA 的吸附载量=（G0—G1）/V。

G0：BSA 总使用量，单位：mg。

G1：偶联反应后未偶联的 BSA 的量，单位：mg。

V：填料的体积，单位：ml。

备注：该偶联方法适用于偶联单抗及其它对较稳定蛋白质类，均能获得较好的偶联效果。

偶联的配体如果是小分子物质（例如氨基酸或其他带-NH2、-SH、-OH 等基团的小分子）配基，则可直接将配体配制成NaOH溶液（10-500mM），根据需要提高偶联的温度，可以大大提高偶联效率。

Key words：

Epoxy-activated Crystarose 6FF