关于上样体积与分离效果的关系

For high resolution fractionation a sample volume from 0.5~4% of the total column volume is recommended, depending on the type of medium used. For most applications the sample volume should not exceed 2% to achieve maximum resolution.

For analytical separations and separations of complex samples, start with a sample volume of 0.5% of the total column volume. Sample volumes less than 0.5% do not normally improve resoulution.

关于上样样品的浓度

To increase the capacity of a gel filtration separation samples can be concentrated. Avoid concentrations above 70 mg/ml protein as viscosity effects may interfere with the separation.

关于流速

Too high a flow rate leads to incomplete partitioning and brand spreading. Conversely, very low flow rates lead to diffusion and band spreading.

分离多聚体

Size exclusion is particularly suited for the resolution of proteins aggregates from monomers. Aggregates are often formed as a result of purification procedures used. Size exclusion is often incorporated as a final polishing step to remove aggregates and act as a buffer-exchange mechanism into the final solution.

使用及维护注意事项

Always use filtered buffers and samples to reduce the need for additional column maintenance.

Always use well degassed buffer to avoid the formation of air bubbles in the packed column during a run.

Buffers, prepacked columns and samples should be kept at the same temperature to prevent air bubbles forming in the column.

Filter cleaning solutions before use and always re-equilibrate the column with 2-3 column volumes of buffer before the next separation.

Superdex柱材料的处理和保存条件

Superdex is stable in all commonly used aqueous buffers, pH 3-12, and additives such as detergents(1% SDS), denaturing agents(8 M urea or 6 M guanidine hydrochloride)

Store unused media +4℃ to 25℃ in 20% ethanol. Do not freeze.

V₀、V_t及V_t - V₀

分子量越大的蛋白越早出峰

常见峰型问题分析

更多内容，请参考：GE Healthcare 的 《Gel Filtration: Principles and methods》

Update

实例Ⅰ：来看下面这个峰型，其原因是上样样品量太大（浓度约60 mg/ml，上样体积1.6 ml）。

相关日志推荐：