实验方法>
免疫学技术>
抗体抗原实验>Protein Kinase A and Signal Transduction in T Lymphocytes: Biochemical and Molecular Methods
Protein Kinase A and Signal Transduction in T Lymphocytes: Biochemical and Molecular Methods
关键词: protein kinase signal来源: 互联网
Abnormal T-cell effector functions in systemic lupus erythematosus (SLE) are present and may be associated with disease immunopathogenesis. Our work has led to the characterization of a signaling defect, involving protein kinase A (PKA), leading to abnormal T-cell effector functions in SLE. PKA is a component of the adenylyl cyclase/cyclic adenosine monophosphate/PKA (AC/cAMP/PKA) pathway, a principal signal transduction system in T cells. The aim of this chapter is to provide a comprehensive, technical, step-by-step approach to studying PKA function in T cells. The methods detailed here are (a) chromatographic fractionation of PKA-I and PKA-II isozymes and PKA phosphotransferase activity in purified T cell populations, (b) Western immunoblotting to identify the presence of regulatory (R)-subunit proteins of PKA, and (c) isolation of RNA, and quantification of PKA R subunit-specific transcripts by competitive polymerase chain reaction. Although our emphasis in the chapter is T cells, these methods may be useful for investigation of signaling via PKA in other cell types as well.
推荐方法
- 流式细胞周期检问答
- 工业微生物产生菌的分离筛选(四)
- 固定和包埋的组织标本DNA PCR反应模板的制备
- SPA技术
- 抗体的稀释度稀释液及稀释度的测定方法
- 条件性基因敲除应用于研究基因的功能
- RNase H: Specificity, Mechanisms of Action, and Antiviral Target
- Production of Monoclonal Antibodies for the Detection of Chemically Modified DNA
- Modelling Emerging Viral Epidemics for Public Health Protection
- Digital Image Analysis of Lymphocyte Activation