分子克隆-蛋白表达实验指南（十一）
SDS-PAGE胶样品排列：
MarkerUII 37CUI’I’
Marker：低分子量蛋白marker，上样10ul
UI：未诱导菌液，上样10ul。任取37C和20C中一个
I：诱导后对照，上样10ul
UI’, I’代表GST等空载体转化的BL21未诱导和诱导后的对照
若表达载体是GST等大分子蛋白tag，则应做一个诱导空载体的对照。SDS-PAGE时取未诱导的空载体和诱导后的空载体一起上样（诱导温度37C，IPTG
1mM），以检测诱导体系是否成功。
18．活性亲和层析
适用蛋白：任何表达在上清的融合蛋白
下面以GST tag为例(GST tag一般需要切除，事先应确认所表达蛋白上没有thrombin等蛋白酶位点)
Column Purification
1. Use a pipet to apply the bacterial sonicate (Procedure 11,page 15) to a
column of drained and washed Glutathione Sepharose 4B RediPack or Disposable
Column (Procedure 8, page 13).
– Note: If needed, the sonicate may be clarified by filtrationthrough a 0.45
µm filter before applying it to the column.
2. Remove the end cap and allow the sonicate to flow through the column.
– Note: The majority of the eluate can be discarded. However, a sample should
be
retained for analysis by SDS-PAGE (see Figure 7; see also Figure 8, page 19)
or CDNB
assay (Procedure 17, page 21) to measure the efficiency of binding to the
matrix.
3. Wash the matrix by the addition of 10 bed volumes* of 1X PBS. Allow the
column to drain. Repeat twice more for a total of three washes.
– Note: Fusion protein bound to the matrix may be eluted directly at this
stage using Glutathione Elution Buffer (see “Column Elution”), or the protein
may be cleaved on the matrix with PreScission Protease, thrombin or factor Xa
to liberate the protein of interest from the GST moiety. [Refer to Procedure
14, PreScission Protease Cleavage (page 17), Procedure 15, Thrombin Cleavage
(page 18), or to Procedure 16, Factor Xa Cleavage (page 19).
4. Once the column with bound protein has been washed and drained, replace the
bottom cap.
5. Elute the fusion protein by the addition of 1 ml of Glutathione Elution
Buffer per ml bed volume. Incubate the column at room temperature (22-25°C)
for 10 minutes to elute the fusion protein.
6. Remove the bottom cap and collect the eluate. This contains the fusion
protein.
7. Repeat the elution and collection steps twice more. Pool the three eluates.
– Note: Following the elution steps, a significant amount of fusion protein
may remain bound to the matrix. Volumes and times used for elution may vary
among fusion proteins. Additional elutions may be required. Eluates should be
monitored for GST fusion protein by SDS-PAGE (see Figure 7, page 16; see also
Figure 8,page 19) or by CDNB assay (Procedure 17, page 21).
