Detection of tyrosine‑phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST‑Nck1‑SH2 
pull‑down and two‑dimensional electrophoresis

Qiu,Fanghua; Huang,Dehong; Xiao,Hongguang; Qiu,Fangying; Lu,Liming; Nie,Jing

doi:10.3892/mmr.2013.1324

April 2013 Volume 7 Issue 4

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April 2013 Volume 7 Issue 4

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Article

Detection of tyrosine‑phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST‑Nck1‑SH2 pull‑down and two‑dimensional electrophoresis

Authors:
- Fanghua Qiu
- Dehong Huang
- Hongguang Xiao
- Fangying Qiu
- Liming Lu
- Jing Nie
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Affiliations: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, P.R. China, Department of Neurology, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, P.R. China, Department of Clinical Laboratory, The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, Guangdong, P.R. China, Department of Emergency, The Fourth Affiliated Hospital of Guangzhou Medical College, Guangzhou, Guangdong, P.R. China, Division of Nephrology, Nanfang Hospital, Southern Medical University, Research Institute of Nephrology Guangdong Province, Key Laboratory for Organ Failure Research, Ministry of Education, Guangzhou, Guangdong, P.R. China
Pages: 1209-1214
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Published online on: February 19, 2013

https://doi.org/10.3892/mmr.2013.1324
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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.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Detection of tyrosine‑phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST‑Nck1‑SH2 pull‑down and two‑dimensional electrophoresis

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