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Quantitative determination of ceramide using human recombinant ceramide kinase
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Quantitative determination of ceramide using human recombinant ceramide kinase

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Introduction Protocol References Credit lines
Category
Glycolipids and related compounds
Protocol Name

Quantitative determination of ceramide using human recombinant ceramide kinase

Authors
Okino, Nozomu *
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.
KeyWords
Reagents

n-octylglucoside (Merck Millipore, Billerica, MA, #494459)

Cardiolipin (Sigma-Aldrich, St. Louis, MO, #C0563)

Diethylenetriaminepentaacetic acid (DETAPAC) (Sigma-Aldrich, #D1133)

Chloroform (CHCl3)

Methanol (MeOH)

CHCl3-MeOH mixture (C/M, volume/volume)

Distilled water (DW)

TLC plate (Silicagel 60, 20 × 20 cm, Merck Millipore)

Instruments

Speed Vac concentrator (Thermo Fisher Scientific Inc., Waltham, MA)

Imaging analyzer (FLA 5000, Fujifilm, Tokyo, Japan)

Dounce homogenizer

Methods
1.

Preparation of the Recombinant Ceramide Kinase

1) 

 Transfect the HEK293 cells with a human CerK expression vector.

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2) 

 Wash the transfected cells with cold phosphate-buffered saline (PBS) after 1 day.

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3) 

 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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4) 

 Disrupt the cells with a Dounce homogenizer.

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5) 

 Centrifuge at 100,000 × g for 60 min.

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6) 

 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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7) 

 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.

Lipid Extraction

1) 

 Harvest the cells (~1 × 106 cells) by centrifugation.

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2) 

 Add 500 μL of MeOH to the sample, and lyse the cells through sonication.

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3) 

 Add 500 μL of CHCl3 and 450 μL of DW.

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4) 

 Centrifuge at 2,000 × g for 5 min.

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5) 

 Dry the organic phase using a Speed Vac concentrator.

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6) 

 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)

1) 

 Transfer the samples (20 μL of solubilized lipid or standard ceramide) to an Eppendorf tube.

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2) 

 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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3) 

 Initiate the reaction by adding 10 μL of [γ-32P]ATP (5 μCi; 10 mM ATP in 100 mM MgCl2).

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4) 

 Incubate at 30°C for 30 min.

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5) 

 Add 0.6 mL of CHCl3/MeOH (1/1, v/v).

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6) 

 Vortex for a few seconds and add 250 μL of 1 M KCl in 20 mM Mops, pH 7.0.

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7) 

 Centrifuge at 10,000 × g for 5 min.

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8) 

 Transfer the organic phase to an Eppendorf tube and dry under a stream of N2 gas.

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9) 

 Dissolve the sample in an aliquot of CHCl3/MeOH (1/1, v/v) and spot on a TLC plate.

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10) 

 Develop the plate with CHCl3/acetone/ MeOH /acetic acid/water (10/4/3/2/1, v/v/v/v/v).

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11) 

 Dry the TLC plate and cover with a wrap.

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12) 

 Expose the TLC plate to an imaging plate.

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13) 

 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.

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Date of registration:2014-07-31 09:58:01
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