Zhou J N, Linder S, Franzén B, Auer G, Hochstrasser D F, Persson M A
Radiumhemmets Research Laboratory, Cancer Center Karolinska, Karolinska Institute and Hospital, Stockholm, Sweden.
Electrophoresis. 1998 Jul;19(10):1808-10. doi: 10.1002/elps.1150191044.
We describe a simple and efficient procedure which can be used to prepare antibodies to proteins extracted by two-dimensional gel electrophoresis (2-DE), using beta-actin as a model. Protein was electroeluted from a stained gel, biotinylated and used for selection of phage from a semisynthetic phage antibody library. After four rounds of selection using 50 ng beta-actin per cycle, approximately 8 X 10(3) phage were recovered. Antibody fragments were prepared from 21 randomly picked clones. Six of eighteen (6/18) antibody-positive clones produced antibody fragments reacting against beta-actin in an enzyme linked immunosorbent assay (ELISA). Sequencing of the HC-CDR3-region showed that all six clones were independent isolates, suggesting that a large number of independent phage antibody reactivities were generated.
我们描述了一种简单高效的方法，该方法可用于制备针对通过二维凝胶电泳（2-DE）提取的蛋白质的抗体，以β-肌动蛋白作为模型。蛋白质从染色凝胶中电洗脱，生物素化后用于从半合成噬菌体抗体库中筛选噬菌体。在每个循环使用50 ngβ-肌动蛋白进行四轮筛选后，回收了约8×10³个噬菌体。从21个随机挑选的克隆中制备抗体片段。在酶联免疫吸附测定（ELISA）中，18个抗体阳性克隆中有6个产生了与β-肌动蛋白反应的抗体片段。重链互补决定区3（HC-CDR3）区域的测序表明，所有6个克隆都是独立分离株，这表明产生了大量独立的噬菌体抗体反应性。
