分子克隆-蛋白表达实验指南（十二）
– Note: The yield of fusion protein can be estimated by measuring the
absorbance at 280 nm. The GST affinity tag can be approximated by 1 A280 Å 0.5
mg/ml. (This is based on the extinction coefficient of the GST monomer using a
Bradford protein assay. Other protein determination methods may result in
different extinction coefficients.).
– Note: The yield of protein may also be determined by standard
chromogenic methods (e.g., Lowry, BCA,Bradford, etc.). If a Lowry or BCA type
method is to be used, the sample must first be dialyzed against 2000 volumes
of 1X PBS to remove glutathione, which can interfere with protein measurement.
The Bradford method can be performed in the presence of glutathione.
Thrombin Cleavage of Fusion Protein Bound to Column/Bulk Matrix
1. Prepare the thrombin reaction mixture as follows: For each ml of
Glutathione Sepharose bed volume*, mix 50(25) μl (U) of thrombin solution and
950 μl of 1X PBS. (When using Thrombin cleavage, 25ul thrombin can be used for
cleavage of 1 ml bed volume. There would be only 0.8mg difference in
concentration of the eluted protein. Thus, for the sake of thrift, 25ul
thrombin enzyme is equally enough. The price for 50U thrombin is 70RMB)
2. Replace the bottom cap on the washed column from Procedure 12, step 6 or
Procedure 13, step 3 and add the Thrombin Protease mixture. If batch format is
used, add Thrombin Protease mixture to Glutathione Sepharose pellet. Gently
shake or rotate the the suspension at room temperature for 2-16 hours. (In the
aim of reducing protein degradation, condition of 10~26h in 4C is recommended
for the complete and thorough digestion of the target protein)
3. Following incubation, remove the bottom cap and collect the eluate in a
clean tube. If batch format is used, centrifuge the suspension at 500 x g for
5 minutes to pellet the Sepharose beads and carefully transfer the eluate to a
clean tube. The eluate will contain the protein of interest while the GST
portion of the fusion protein remains bound to the Glutathione Sepharose
matrix.
19. 割胶回收
预先配制20cm×20cm的SDS-PAGE胶，并且割胶前先提前一天跑一块相同浓度的SDS-PAGE小胶，用于计算大胶上目的蛋白的比例。
适用蛋白：表达在IB中，tag为大分子蛋白，如GST等。
1． 用不同浓度梯度（1M, 2M, 4M,
8M）的urea缓冲液洗涤包涵体，除去多余的杂蛋白，割胶时能减少杂蛋白的共纯化。Urea梯度选择能保留目的蛋白于IB中的最大浓度。
2． 用step1摸好的urea条件重悬超声离心后的IB，vortex使充分溶解。
3． 12000rpm, 10min,4C，去除不溶于urea的细胞杂质。
4． 取4－5ml离心后的上清，加入SDS-PAGE loading buffer至1×。沸水浴5min（最好将其分装于1.5ml
EP管中，使水浴时充分受热）。其余-20C保存。
5． 水浴后上样，200V，3－4h至溴芬蓝染料走到SDS-PAGE胶底。
6．
取5－7cm的半透膜，沸水浴5min，之后放入蛋白电泳buffer中。电透析tube和frit也放入buffer中。浸泡5min除去半透膜，frit，tube上的微小气泡后，将frit放入tube带磨沙的开口中，深入大约0.2～0.5cm（若插入太高，会导致最终电透析后的蛋白浓度较低，不满足打抗体的1mg/ml浓度）。之后小心的将半透膜套在放了frit的tube开口上，用橡皮筋将半透膜套紧。尽量杜绝frit和半透膜之间的气泡，它会影响透析的效率。
