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Assay of glucocerebrosidases using HPLC and fluorescent substrates
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Assay of glucocerebrosidases using HPLC and fluorescent substrates

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

Assay of glucocerebrosidases using HPLC and fluorescent substrates

Authors
Hayashi, Yasuhiro *
Faculty of Pharmaceutical Sciences, Teikyo 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

C6-NBD-GlcCer (#N6783: Sigma-Aldrich, St. Louis, MO)

C12-BODIPY-GlcCer (#D7547: Invitrogen/Life Technologies, Carlsbad, CA)

Normal-phase column (Intersil SIL 15A-5: GL-Sciences, Tokyo, Japan)

Isopropyl alcohol

n-hexane

Instruments

HPLC (L-7100: Hitachi, Ltd., Tokyo, Japan)

Fluorescent detector (L-7480: Hitachi, Ltd., Tokyo, Japan)

Autosampler (L-7200: Hitachi, Ltd., Tokyo, Japan)

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

Methods
1.

Assay of glucocerebrosidases using HPLC and fluorescent substrates

1) 

 For the AGC assay, 20 μL of reaction mixture should contain 0.6% sodium taurocholate, 0.25% Triton X-100, and 2.5 mM C6-NBD-GlcCer or 0.25 mM C12-BODIPY-GlcCer in 50 mM phosphate-citrate buffer, pH 5.0. For the NGC assay, 20 μL of reaction mixture should contain 0.25% sodium cholate, 1 mM CBE, and 2.5 mM C6-NBD-GlcCer in 50 mM Hepes buffer, pH 7.0.

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2) 

 Stop the reaction by adding 200 μL of chloroform/methanol (2:1, v/v). After vortexing for a few seconds, centrifuge the reaction mixture.

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3) 

 Dry the organic phase, dissolve lipids in 200 μL of isopropyl alcohol/n-hexane/H2O (55:44:1, v/v/v), and then transfer to a glass vial in an auto-sampler (HITACHI L-7200).

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4) 

 Determine fluorescence using a detector set to excitation and emission wavelengths of 470 and 530 nm for the assay with C6-NBD-GlcCer, and 505 and 540 nm for the assay with C12-BODIPY-GlcCer, respectively.

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5) 

 An aliquot of sample is automatically loaded onto a normal-phase column and eluted with isopropyl alcohol/n-hexane/H2O (55:44:1, v/v/v) for the assay using C6-NBD-GlcCer as a substrate or isopropyl alcohol/n-hexane/H2O (55:85:51, v/v/v) for that using C12-BODIPY-GlcCer as a substrate at a flow rate of 2.0 mL/min.

Comment 0
Notes

With this procedure, 50 fmol – 50 pmol of fluorescent Cer released from fluorescent GlcCer can be determined.

Figure & Legends

Figure & Legends

 

Reprinted from Anal Biochem., 383(1), Hayashi Y, Ito M. et al., A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral beta-glucocerebrosidases, 122-9, 2008, with permission from Elsevier. doi:10.1016/j.ab.2008.07.024.

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