SDS-PAGE膠樣品排列：
MarkerUII 37CUI’I’
Marker：低分子量蛋白marker，上樣10ul
UI：未誘導菌液，上樣10ul。任取37C和20C中一個
I：誘導后對照，上樣10ul
UI’, I’代表GST等空載體轉化的BL21未誘導和誘導后的對照
若表達載體是GST等大分子蛋白tag，則應做一個誘導空載體的對照。SDS-PAGE時取未誘導的空載體和誘導后的空載體一起上樣（誘導溫度37C，IPTG
1mM），以檢測誘導體系是否成功。
18．活性親和層析
適用蛋白：任何表達在上清的融合蛋白
下面以GST tag為例(GST tag一般需要切除，事先應確認所表達蛋白上沒有thrombin等蛋白酶位點)
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).