– 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. 割膠回收
預先配制20cm×20cm的SDS-PAGE膠，并且割膠前先提前一天跑一塊相同濃度的SDS-PAGE小膠，用于計算大膠上目的蛋白的比例。
適用蛋白：表達在IB中，tag為大分子蛋白，如GST等。
1． 用不同濃度梯度（1M, 2M, 4M,
8M）的urea緩沖液洗滌包涵體，除去多余的雜蛋白，割膠時能減少雜蛋白的共純化。Urea梯度選擇能保留目的蛋白于IB中的最大濃度。
2． 用step1摸好的urea條件重懸超聲離心后的IB，vortex使充分溶解。
3． 12000rpm, 10min,4C，去除不溶于urea的細胞雜質。
4． 取4－5ml離心后的上清，加入SDS-PAGE loading buffer至1×。沸水浴5min（最好將其分裝于1.5ml
EP管中，使水浴時充分受熱）。其余-20C保存。
5． 水浴后上樣，200V，3－4h至溴芬藍染料走到SDS-PAGE膠底。
6．
取5－7cm的半透膜，沸水浴5min，之后放入蛋白電泳buffer中。電透析tube和frit也放入buffer中。浸泡5min除去半透膜，frit，tube上的微小氣泡后，將frit放入tube帶磨沙的開口中，深入大約0.2～0.5cm（若插入太高，會導致最終電透析后的蛋白濃度較低，不滿足打抗體的1mg/ml濃度）。之后小心的將半透膜套在放了frit的tube開口上，用橡皮筋將半透膜套緊。盡量杜絕frit和半透膜之間的氣泡，它會影響透析的效率。