Pulification and identification of substrate proteins for selective ubquitin-dependent proteolysis during NGF-induced neurite outgrowth of PC12h cells.

优化纯化协议.使用我们为纯化细胞ubiquitinated蛋白建立的K562细胞方法。第一步是将免疫亲和性色谱法应用到单克隆抗体FK 2到ubiquitin。1毫升的FK 2-Sepharose凝胶可以吸收0.2毫克的泛素等价物，该吸收材料完全用3..。5 M MgCl型D22型D2。然后，材料用Superdex 75进行凝胶过滤，两个特征片段产生了高分子权重（HMW） （>30 kDa）和低分子权重（LMW） （<30 kDa）。该配方主要由多ubiquitinated蛋白质组成。在我们最近发现了用ubiquitin-jugating Enzymes的ubiquitin-thiosters（E2）作为ubiquitin-thiosters.在神经胶质细胞中发现的ubiquitin-thiosters.通过免疫吸收使用FK 2抗体和SDS-PAGE的组合，我们在PC 12 h细胞中可视化的ubiquitin-thiosters在其存在中被培养的蛋白质。 ... More worth factor （NGF）。三个波段， 18、19和36-kDa，最近出现的六个波段的强度， 20、23、26、51、68和93-kDa，增加了无处不在的蛋白质，无处不在的蛋白质混合物（0.56毫克蛋白质）通过免疫亲和性色谱法获得了经过NGF-treatment的PC 12 h细胞（2x 10-D19-D1细胞）。混合物进一步由凝胶过滤分离，并在HMW和LMW的两个分区中得到结果。几个蛋白质带从SDS-PAGE的latter中分离出来，以及由Lysyl-endopeptida捕获的肽片段的氨基酸序列在逆转中生长。在ubiquitin digests和five unknown sequences中的两个氨基酸序列的分析。A monoclonal antibody against ubiquitin C-terminal tag peptide。此抗体是必需标识的target proteins。因为抗原的不相容性，我们必须改变肽的设计，并最终确定了所希望的抗体，因此可以应用于对HMW不含蛋白的分析。Less（低）

Optimization of purification protocols.Using K562 cells we established methods for purifying cellular ubiquitinated proteins. The first step is an immunoaffinity chromatography using a monoclonal antibody FK2 to ubiquitin. One ml of FK2-Sepharose gel was able to adsorb abount 0.2 mg of ubiquitin equivalent, and the adsorbed materials were completely eluted with 3..5 M MgClィイD22ィエD2. Then the materials were fractionated by gel filtration using Superdex 75, and two characteristic fractions having high molecular weights (HMW) (>30 kDa) and low molecular weights (LMW) (<30 kDa) were obtained. The former was mainly composed of multi-ubiquitinated proteins. In the latter we found ubiquitin thioesters with ubiquitin-conjugating enzymes (E2) as well as ubiquitinated proteins.Ubiquitinated protein variety during neurite elongation.By the combination of immunoprecipitation using FK2 antibody and SDS-PAGE, we visualized ubiquitinated proteins in PC12h cells cultivated in the presence of nerve gro … More wth factor (NGF). Three bands, 18, 19, and 36-kDa, newly appeared, and intensities of six bands, 20, 23, 26, 51, 68, and 93-kDa, increased.Purification of ubiquitinated proteins.Ubiquitinated protein mixtures (0.56 mg protein) were obtained from NGF-treated PC12h cells (2 x 10ィイD19ィエD1 cells) by the immunoaffinity chromatography. The mixtures were further separated by the gel filtration, and resulted in the two distinctive fractions of HMW and LMW. Several protein bands were isolated from the latter by SDS-PAGE, and amino acid sequencing of their peptide fragments obtained by lysyl-endopeptidase is ongoing in turns. The analysis of the 26-kDa band resulted in two amino acid sequences of ubiquitin digests and five unknown sequences.A monoclonal antibody against ubiquitin C-terminal tag peptide.This antibody is required for identification of the target proteins. Because of antigen insolubility, we had to change the peptide design, and finally established the desired antibody, which will be applied to the analysis of HMW ubiquitinated proteins. Less