title: Detection of tyrosine‑phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST‑Nck1‑SH2 pull‑down and two‑dimensional electrophoresis.
abstract: Tyrosine-phosphorylated proteins govern a host of cell functions, such as growth, division, adhesion and motility. We previously identified a group of Nck Src homology 2 (SH2) domain‑binding proteins by combining the GST-Nck1-SH2 pull-down method with two-dimensional electrophoresis (2‑DE) in hepatocellular carcinoma (HCC) tissues. In the present study, different methods and conditions for key procedures of GST-Nck1-SH2 pull-down and 2‑DE were investigated and optimized. High-resolution results were obtained using the following conditions: a total amount of 100 µl GST-Nck1-SH2 fusion proteins/10 mg liver proteins to execute the pull-down procedure; 7 M urea and 2 M thiourea as lysis buffer; ultrafiltration depletion of interferential materials. Moreover, we performed a negative control experiment using GST-4T3 during the pull-down procedure, and further demonstrated that the proteins obtained using the aforementioned method interacted with Nck in a tyrosine phosphorylation-dependent manner. The optimized method offers a rapid, efficient alternative for the high‑quantity screening of tyrosine-phosphorylated protein expression and solubility, which in turn facilitates future studies on tyrosine-phosphorylated proteins.
authors: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, P.R. China.
authors: Fanghua
authors: F
authors: Qiu
authors: Dehong
authors: D
authors: Huang
authors: Hongguang
authors: H
authors: Xiao
authors: Fangying
authors: F
authors: Qiu
authors: Liming
authors: L
authors: Lu
authors: Jing
authors: J
authors: Nie
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D002465:Cell Movement / Q000235:genetics; D004586:Electrophoresis; D005786:Gene Expression Regulation; D006498:Hepatectomy; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000601:surgery; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015513:Oncogene Proteins; D027361:Organic Anion Transporters; D014443:Tyrosine; C078332:Nck protein; C487009:SLCO6A1 protein, human