文档介绍：实验十凝胶过滤柱层析纯化 碱性磷酸酶
一、目的要求
。
本次实验安排
部分操作步骤介绍
分组,各组装柱、平衡
继续介绍原理、步骤及仪器的使用
各组上样、分离蛋白质(仪器调节好后,可以离开吃饭,各组留一个同学观察)
吃完饭收集样品,测定酶活力,整理玻璃仪器,结束实验。
二、原理
凝胶过滤层析也称分子筛层析、排阻层析。是利用具有网状结构的凝胶的分子筛作用,根据被分离物质的分子大小不同来进行分离。
AGAROSE琼脂糖凝胶
Gel structure
1. Spherical particles packed into a column
2. Sample applied
3. Buffer mobile phase and sample move through column, molecules diffuse in and out of matrix
4. large molecules leave the column first followed by smaller molecules in order of size, molecules larger than the matrix pores pass straight through.
Diffusion
Buffer
Diffusion into the pores
Diffusion out of the pores
Buffer
Steric exclusion
Steric exclusion leads to early elution
Molecules elute in order of size.
The largest molecules elute first; the smallest ones elute last.
内水体积Vi
外水体积Vo
柱床体积Vt
洗脱体积Ve
Vt=Vo+Vi+Vg
Ve=Vo+KdVi
Kd=(Ve-Vo)/Vi
Vo = void volume
Ve elution volume
Ve2= total liquid volume= Vo+Vi
Vi = inner pore volume = Vt - Vs - Vo
Vt = total geometric volume of the column
void volume Vo
volume of the
gel matrix Vs
pore volume Vi
Vo
VE1
VE2
Vt