Electrophoresis of proteins and protein-protein complexes in a native agarose gel
Electrophoresis of proteins and protein-protein complexes in a native agarose gel
复制标题
DOI：
10.1006/abio.2000.4598
复制
发表时间：
2000-06-15
影响因子：
2.9
通讯作者：
Kim, SH
中科院分区：
文献类型：
--
作者：
Kim, R;Yokota, H;Kim, SH
材料和方法。琼脂糖（低电内渗）购自Research Organics（Cleveland，OH）。在我们的实验室中纯化了以下蛋白质：单链莫内林（SCM）（3），詹氏甲烷球菌小热休克蛋白（Mj HSP 16）。5）（4）和Mj 0577蛋白（5）、谷胱甘肽S-转移酶和Pyrococcus horikoshii 1704（Ph 1704）。牛血清白蛋白购自Sigma（St. Louis，MO）。分枝如Zarembinski等人所述制备詹氏细胞提取物。（五）、使用浸没式水平Gibco BRL Horizon 58单元进行天然琼脂糖电泳。在缓冲液A（25 mM Tris-HCl，pH 8.5，19.2 mM甘氨酸）中制备水平的0.8%琼脂糖凝胶（8 cm 5.5 cm 3 mm），并将梳状物置于凝胶中心。将凝胶浸没在含有缓冲液A的储器中，并在室温下以50 V的恒定电压进行电泳1小时。上样前，将样品（5 μg）与样品缓冲液（20%甘油、0.2%溴酚蓝、0.12 M Tris碱）以1：1混合。将凝胶在0.12%考马斯亮蓝R、45%甲醇、10%乙酸中染色20分钟，并在45%甲醇、10%乙酸中脱色，并在两层玻璃纸膜（Idea Scientific，Minneapolis，MN）之间干燥。为了分离蛋白质：蛋白质复合物的组分，将两个相同的样品上样到相邻泳道上。通过对含有复合物的两条泳道之一染色并使用该信息来确定复合物的位置;用剃刀刀片切下未染色的凝胶片段并置于凝胶喷雾器中，该凝胶喷雾器插入Micropure-0.22单元（Millipore Corp.，贝德福德，马萨诸塞州）（2）。将这两个单元在12，000 g下离心，使凝胶通过喷雾器的孔，从而将凝胶转化为细浆。然后通过Micropure-0.22捕获凝胶颗粒，并将可溶性生物分子挤出并通过Micropure-0.22。
Materials and methods. Agarose (low electroendosmosis) was purchased from Research Organics (Cleveland, OH). The following proteins were purified in our laboratory: single-chain monellin (SCM)(3), Methanococcus jannaschii small heat shock protein (Mj HSP16. 5)(4) and Mj0577 protein (5), glutathione S-transferase, and Pyrococcus horikoshii 1704 (Ph1704). Bovine serum albumin was purchased from Sigma (St. Louis, MO). The M. jannaschii cell extract was prepared as described by Zarembinski et al.(5). Native agarose electrophoresis was carried out using a submerged horizontal Gibco BRL Horizon 58 unit. The horizontal 0.8% agarose gel (8 cm 5.5 cm 3 mm) was prepared in Buffer A (25 mM Tris–HCl, pH 8.5, 19.2 mM glycine) and the comb placed in the center of the gel. The gel was submerged in a reservoir containing Buffer A and electrophoresis was performed at a constant voltage of 50 V for 1 h at room temperature. The samples (5 μg) were mixed 1: 1 with sample buffer (20% glycerol, 0.2% Bromophenol blue, 0.12 M Tris base) prior to loading. Gels were stained in 0.12% Coomassie brilliant blue R, 45% methanol, 10% acetic acid for 20 min and destained in 45% methanol, 10% acetic acid and dried between two layers of cellophane membrane (Idea Scientific, Minneapolis, MN). To isolate the components of a protein: protein complex, two identical samples were loaded on adjacent lanes. The position of the complex was determined by staining one of the two lanes containing the complex and using this information; the unstained gel fragment was cut out with a razor blade and placed into a gel nebulizer that was inserted into a Micropure-0.22 unit (Millipore Corp., Bedford, MA)(2). These two units were centrifuged at 12,000 g, allowing the gel to go through the orifice of the nebulizer thus converting the gel to a fine slurry. The gel particles were then captured by the Micropure-0.22 and the soluble biomolecules were extruded and passed through the Micro-