7．
電泳結束后，按比例從膠上割下相應約1cm條帶（當按比例的條帶割下后可相應的向兩邊再割一點，但是電透析時中間和兩邊的膠必須分開透析），用鑷子或尺子將膠碾碎成2mm見方的小塊。
8． 將碎塊小心加入電透析tube中，200V，120～150min。
9． 移出放電透析tube的架子，用注射器將tube上部的buffer吸掉。之后小心的在下部半透膜上插入注射器針頭，緩緩析出其中的蛋白溶液。
10．吸取的蛋白溶液轉入1.5ml EP管中，取20ul用于SDS-PAGE純度驗證，其余的-20C保存。
割膠的比例計算：
小膠（染色后）： 大膠（未染色）：
分離膠頂部――――― 分離膠頂部―――――
A cm
目的蛋白位置―――― B cm C cm 目的蛋白位置？
溴芬藍底部位置――― 溴芬藍底部位置――――
簡單的比例計算即可得到大膠上目的蛋白的位置。
20．純化后蛋白SDS-PAGE驗證純度
SDS-PAGE方法如前所述，該結果必須拍照存檔, 格式如下：
Lane1～5：Marker、未誘導、誘導后、上清、沉淀
Lane6～9：純化后蛋白依次按1.5, 0.75, 0.375, 0.187mg/ml上樣，10ul each lane.
當lane 8看不到雜帶時，蛋白純度才符合要求
21. 其他常用操作及知識
SDS-PAGE：
蛋白質如果能融解在SDS溶液中，則它們可以按分子質量的大小加以分離。這種方法的原理為小分子質量的SDS與蛋白質的疏水基團結合，破壞了蛋白質的層疊結構，使它以舒展態穩定地存在于溶液中，每克蛋白質約結合1.4gSDS。SDS分子以它自己地負電荷掩蓋了蛋白質分子原來存在的電荷，各種蛋白質分子表現出的電荷密度相等，SDS-蛋白質復合物的長度與其分子質量成比例，電泳時，它們純粹按照分子量的大小由凝膠的分子篩效應進行分離。
層析柱的再生
Regeneration of Ni-NTA beads(from QIAGEN):
Handling
Ni-NTA matrices are stable under a wide variety of conditions and need not be
refrigerated, except to inhibit growth of microorganisms for long-term
storage. After use they should be washed for 30 minutes with 0.5M NaOH. Ni-NTA
matrices should be stored in 30% ethanol to inhibit microbial growth. The
matrix can be stored for up to one week in any of the denaturing buffers.
Reuse of Ni-NTA Resin
The reuse of Ni-NTA resin depends on the nature of the sample and should only
be performed with identical recombinant proteins. Based on the experience of
Hoffmann-La Roche Ltd. (Basel, Switzerland), who have purified more than 100
different proteins on Ni-NTA resin, we recommend a maximum of 5 runs per
column.
If the Ni-NTA Agarose changes from light blue to brownish-gray, the following
regeneration procedure is recommended.
Procedure:
1. Wash the column with 2 volumes of Regeneration Buffer (6 M GuHCl, 0.2 M
acetic acid).
2. Wash the column with 5 volumes of H2O.
3. Wash the column with 3 volumes of 2% SDS.
4. Wash the column with 1 volume of 25% EtOH.
5. Wash the column with 1 volume of 50% EtOH.
6. Wash the column with 1 volume of 75% EtOH.
7. Wash the column with 5 volumes of 100% EtOH.
8. Wash the column with 1 volume of 75% EtOH.
9. Wash the column with 1 volume of 50% EtOH.
10. Wash the column with 1 volume of 25% EtOH.
11. Wash the column with 1 volume of H2O.
12. Wash the column with 5 volumes of 100 mM EDTA, pH 8.0.
13. Wash the column with H2O.
14. Recharge the column with 2 volumes of 100 mM NiSO4.
15. Wash the column with 2 volumes of H2O.
16. Wash the column with 2 volumes of Regeneration Buffer.
17. Equilibrate with 2 volumes of a suitable buffer (e.g., Buffer A or B).
Regeneration of Glutathione Sepharose 4B(GST)
Glutathione Sepharose 4B may be regenerated for re-use by washing the gel with
2-3 bed volumes(Bed volume is equal to 0.5 x the volume of the 50% Glutathione
Sepharose slurry used or 0.75 x the volume of the originalGlutathione
Sepharose slurry) of alternating high pH (0.1 M Tris-HCl + 0.5 M NaCl, pH 8.5)
and low pH(0.1 M sodium acetate + 0.5 M NaCl, pH 4.5) buffers. This cycle
should be repeated 3 times followed by re-equilibration with 3-5 bed volumes
of 1X PBS.
If the gel appears to be losing binding capacity, it may be due to an
accumulation of precipitated, denatured or non-specifically bound proteins.
To remove precipitated or denatured substances, wash the matrix with 2 bed
volumes of 6 M guanidine hydrochloride, immediately followed by a wash with 5
bed volumes of 1X PBS.
To remove hydrophobically bound substances, wash the matrix with 3-4 bed
volumes of 70% ethanol or with 2 bed volumes of a non-ionic detergent (conc.
0.1%), immediately followed by a wash with 5 bed volumes of 1X PBS.
For long-term storage (>1 month), the following procedure of additional washes
is recommended:
1. Wash the gel twice with 10 bed volumes of 1X PBS.
2. Repeat washes using 20% ethanol.
3. Store at +4°C.
4. Re-equilibrate the gel with 1X PBS before re-use.
