A gel-electrophoretic analysis for improved sensitivity and specificity of DNA-dependent protein kinase activity.
A gel-electrophoretic analysis for improved sensitivity and specificity of DNA-dependent protein kinase activity.
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凝胶电泳分析可提高 DNA 依赖性蛋白激酶活性的灵敏度和特异性。
DOI：
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发表时间：
1999
影响因子：
2
通讯作者：
K. Hirano
中科院分区：
文献类型：
--
作者：
Y. Matsumoto;N. Umeda;N. Suzuki;K. Sakai;K. Hirano
DNA依赖蛋白激酶(DNA-PK)被认为是DNA双链断裂修复和/或信号转导过程中的关键酶。DNA-PK活性的测定主要通过以合成肽为底物的DNA加减法或DNA下拉法，然后进行滤膜结合分析，即与磷酸纤维滤膜结合的酸不溶放射性的液体闪烁计数。考虑到滤膜结合酸不溶计数中其他细胞蛋白的非特异性磷酸化可能会干扰多肽底物特异性磷酸化的检测，我们通过反应混合物的SDS凝胶电泳法检测了这些检测方法的特异性和特点。电泳图谱显示，除特定底物外，许多非特异性蛋白条带都发生了磷酸化。小鼠L5178Y或FSA1233细胞的DNA-PK活性很低，底物带计数明显。即使在DNA-PK从大多数其他蛋白激酶中分离出来的DNA-PK程序之后，其他细胞蛋白的大量磷酸化仍然受到污染。因此，通过选择性地计数特定的条带，可以可靠地定量小量的多肽底物的特异性磷酸化。这些结果表明，过滤结合分析的DNA-PK活性可能被其他细胞蛋白的非特异性磷酸化所污染，凝胶电泳法将提高合成肽底物DNA-PK的特异性磷酸化的可检测性，从而在灵敏度和特异度上都将提高。
DNA-dependent protein kinase (DNA-PK) is considered a critical enzyme in the repair and/or signal transduction of DNA double-strand breaks. DNA-PK activity has been mostly measured through "DNA-plus-minus" or "DNA-pull-down" procedures using synthetic peptide as substrate followed by filter-binding analysis, i.e. liquid scintillation counting of acid-insoluble radioactivity bound to phosphocellulose filter. Considering that non-specific phosphorylation of other cellular proteins in filter-bound acid-insoluble count could interfere with the detection of specific phosphorylation of peptide substrate, we examined the specificity and characteristics of these assay procedures by SDS gel-electrophoresis of the reaction mixture. The electrophoretic pattern showed phosphorylation in wide range of non-specific protein bands other than the specific substrate. The very low DNA-PK activity shown by murine L5178Y or FSA1233 cells was unambiguously detectable as the count in substrate band. Even following DNA-pull-down procedure, which would separate DNA-PK from most of other protein kinases, substantial amount of phosphorylation of other cellular proteins were still contaminated. Thus by selectively counting the particular bands, small amount of specific phosphorylation of peptide substrate was reliably quantified. These results indicated that the DNA-PK activity through filter-binding analysis was, as suspected, contaminated by non-specific phosphorylation of other cellular proteins and also that the gel-electrophoretic analysis would improve detectability of specific phosphorylation by DNA-PK of synthetic peptide substrate and, therefore, would improve the kinase assay in both sensitivity and specificity.
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影响因子：
56.9
作者：
TACCIOLI, GE;GOTTLIEB, TM;JEGGO, PA
通讯作者：
JEGGO, PA
影响因子：
56.9
作者：
Canman, CE;Lim, DS;Siliciano, JD
通讯作者：
Siliciano, JD
影响因子：
10.5
作者：
Tibbetts, RS;Brumbaugh, KM;Abraham, RT
通讯作者：
Abraham, RT
影响因子：
56.9
作者：
Banin, S;Moyal, L;Ziv, Y
通讯作者：
Ziv, Y