highly specific antibodies for co-detection of human choline kinase α1 and α2 isoforms高度的特定抗体co-detection人类胆碱激酶α1和α2亚型.pdfVIP

Highly Specific Antibodies for Co-Detection of Human Choline Kinase a1 and a2 Isoforms 1,2 1 1 2 Wei Cun See Too *, Mun Teng Wong , Ling Ling Few , Manfred Konrad 1 School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia, 2 Max Planck Institute for Biophysical Chemistry, Goettingen, Germany Abstract Background: Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKa1, a2, and b). Specific inhibition of CKa has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKa used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the a1 or the a2 isoform. Methodology/Principal Findings: In this study, an antiserum against CKa was produced by immunizing rabbits with denatured, purified recombinant CKa2 full-length protein. This antiserum was highly specific for CKa when tested with extracts from different cell lines, and there was no cross reactivity with purified CKb and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKa1 and a2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKa in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKa was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CK