Removal of nonspecific binding proteins is required in co-immunoprecipitation with nuclear proteins
Xi Zeng,
Wei-Hong Zeng,
Jun Zhou,
Xiao-Min Liu,
Guo Huang,
Hong Zhu,
Shuai Xiao,
Ying Zeng,
Deliang Cao
Affiliations
Xi Zeng
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Wei-Hong Zeng
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Jun Zhou
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Xiao-Min Liu
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Guo Huang
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Hong Zhu
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Shuai Xiao
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Ying Zeng
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Deliang Cao
1Hunan Province Key Laboratory of Tumor Cellular & Molecular Pathology, Cancer Research Institute, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China
Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the decrease of ion concentrations but reduced by Nonidet P40. Western blot indicated that p65 and β-actin were present as nonspecifically bound protein to the beads. p53 and β-actin were present in the CO-IP precipitates of nuclear proteins but pretreatment cleared the nonspecifically pulled down p53 and β-actin. These data suggest that magnetic beads are better for CO-IP, but preclearing is necessary to minimize false positive regardless of which bead is used, particularly for nuclear proteins.
