With this method, a human recombinant ceramide kinase (hCerK) is used instead of DGK for the quantification of ceramide in biological samples. Ceramide is phosphorylated with [32P] by hCerK in the presence of [γ-32P]ATP. The radio-labeled ceramide-1 phosphate ([32P]Cer-1-P) thus obtained is separated by TLC and determined with an imaging analyzer. |
Category | Glycolipids and related compounds |
Protocol Name | Quantitative determination of ceramide using human recombinant ceramide kinase |
Authors
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Okino, Nozomu
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Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
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KeyWords |
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Reagents
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n-octylglucoside (Merck Millipore, Billerica, MA, #494459) |
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Cardiolipin (Sigma-Aldrich, St. Louis, MO, #C0563) |
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Diethylenetriaminepentaacetic acid (DETAPAC) (Sigma-Aldrich, #D1133) |
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CHCl3-MeOH mixture (C/M, volume/volume) |
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TLC plate (Silicagel 60, 20 × 20 cm, Merck Millipore) |
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Instruments
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Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA) |
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Imaging analyzer (FLA 5000, Fujifilm, Tokyo, Japan) |
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Methods |
1. |
Preparation of the Recombinant Ceramide Kinase
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1) |
Transfect the HEK293 cells with a human CerK expression vector. |
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Wash the transfected cells with cold phosphate-buffered saline (PBS) after 1 day. |
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Scrape the cells in lysis buffer (20 mM Mops, pH 7.2, containing 2 mM EGTA, 1 mM DTT, 10% glycerol, and protease inhibitor cocktail). |
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Disrupt the cells with a Dounce homogenizer. |
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Centrifuge at 100,000 × g for 60 min. |
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Re-suspend the precipitated membrane fraction in the lysis buffer described above, flash freeze, and store in aliquots at −80°C. |
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Determine the specific activity of the ceramide kinase (CerK) using a ceramide standard. One unit of the CerK is defined as the amount capable of generating 1 pmol of Cer-1-P per minute under the conditions described below. |
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2. |
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Harvest the cells (~1 × 106 cells) by centrifugation. |
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Add 500 μL of MeOH to the sample, and lyse the cells through sonication. |
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Add 500 μL of CHCl3 and 450 μL of DW. |
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Centrifuge at 2,000 × g for 5 min. |
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Dry the organic phase using a Speed Vac concentrator. |
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Re-suspend the dried sample by brief sonication in 20 μL of 10 mM imidazole, pH 6.6, containing 7.5% of n-octylglucoside, 5 mM cardiolipin, and 0.2 mM diethylenetriaminepentaacetic acid (DETAPAC). |
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3. |
Measurement of Ceramide Content Using Ceramide Kinase (CerK)
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1) |
Transfer the samples (20 μL of solubilized lipid or standard ceramide) to an Eppendorf tube. |
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Mix with 60 μL of 20 mM Mops buffer, pH 7.0, containing 50 mM NaCl, 1 mM dithiothreitol, 3 mM CaCl2 and 10 μL of recombinant hCerK (20 μg, 25 units). |
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Initiate the reaction by adding 10 μL of [γ-32P]ATP (5 μCi; 10 mM ATP in 100 mM MgCl2). |
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Add 0.6 mL of CHCl3/MeOH (1/1, v/v). |
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Vortex for a few seconds and add 250 μL of 1 M KCl in 20 mM Mops, pH 7.0. |
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Centrifuge at 10,000 × g for 5 min. |
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Transfer the organic phase to an Eppendorf tube and dry under a stream of N2 gas. |
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Dissolve the sample in an aliquot of CHCl3/MeOH (1/1, v/v) and spot on a TLC plate. |
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Develop the plate with CHCl3/acetone/ MeOH /acetic acid/water (10/4/3/2/1, v/v/v/v/v). |
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Dry the TLC plate and cover with a wrap. |
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Expose the TLC plate to an imaging plate. |
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Determine the radioactivity of the bands corresponding to [32P]Cer-1-P with an imaging analyzer. |
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Discussion | With this procedure, 5–1000 pmol of ceramide can be determined. The method seems to be more sensitive than that using DGK. However, CerK is not commercially available at present. |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-07-31 09:58:01 |
- Bektas, M., Jolly, P.S., Milstien, S., and Spiegel, S. (2003) A specific ceramide kinase assay to measure cellular levels of ceramide. Anal Biochem. 320, 259–265 [PMID : 12927832]
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Ito, Makoto,
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Quantitative determination of ceramide using human recombinant ceramide kinase.
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Ito, Makoto,
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