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Ceramidase activity assay

RX Ruijuan Xu
KW Kai Wang
IM Izolda Mileva
YH Yusuf A. Hannun
LO Lina M. Obeid
CM Cungui Mao
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Total membranes were isolated from cells and were assayed for ceramidase activity assays with different ceramides or ceramide analogs as substrates as described in our previous study [54]. Briefly, acid ceramidase activity encoded by ASAH1 was assayed at pH 4.5 using D-e-C12-ceramide as a substrate. Neutral ceramidase activity encoded by ASAH2 was assayed at pH 7.4 using D-e-C12-NBD as a substrate in the absence of Ca2+. Alkaline ceramidase activity encoded by ACER1, ACER2, or ACER3 was assayed at pH 8.5 in the presence of 5 mM Ca2+ using D-e-C12-NBD-ceramide, D-e-C16-ceramide, or D-ribo-C12-NBD-ceramide, respectively, as a substrate. SPH released from regular ceramides was determined by LC-MS/MS and NBD-fatty acid released from D-e-C12-NBD-ceramide or D-ribo-C12-NBD-ceramide was determined by thin liquid chromatography (TLC).

Copyright and License information:  ©2016 Xu et al.
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