Extraction of the SecinH3 from mouse liver

doi:10.1038/nprot.2006.414

Method Article

Extraction of the SecinH3 from mouse liver

https://doi.org/10.1038/nprot.2006.414

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Biological techniques

Biochemistry

Computational biology and bioinformatics

SecinH3

mouse liver

high performance liquid chromatography (HPLC)

mass spectrometry

Mice fed with the cytohesin inhibitor SecinH3 for two days develop hepatic insulin resistance that can be identified by reduced liver glycogen levels, increased serum insulin and ketone body levels and decreased serum non-esterified fatty acid. To confirm the presence and identity of SecinH3 in mouse liver, we extracted the compound from liver homogenates with chloroform and identified it by LC/MS.

ca. 300 mg mouse liver

chloroform

methanol

PBS

HPLC/MS: Agilent 1100 Series HPLC with Bruker Daltonics Esquire Series MS

HPLC columns:

10x4.0 mm with Nucleosil RP18 5 µm
125x2.0 mm with MultoHigh RP18 5 µm

Polytron (Kinematica)

SpeedVac

300 mg mouse liver each weighed into 15 mL centrifugation tubes and taken up in 1 ml PBS, 2 mL chloroform added
mouse liver homogenized by polytron
incubated at RT for 30’ under agitation,
Centrifuged 5’ at maximum speed
Chloroform phase transferred into fresh centrifugation tube
Water phase extracted second time on shaker with 1.5 mL chloroform, 20’
Centrifuged 5’ at maximum speed
Chloroform phases combined and solvent removed on SpeedVac
Residue dissolved in 100 µL/(100 mg liver) methanol, centrifuged 5’ at 14.000 rpm and liquid transferred into HPLC glass vial
Fractionated via HPLC on Nucleosil RP18 material 5 µm (10x4.0 mm column); gradient: 0-7 min: 70% water, 30% methanol

7-15 min: 40% water, 60% methanol

15-20 min: 100% methanol;

fractions 9-12 min collected

solvent of collected fractions removed by SpeedVac and residue taken up in 50 µl methanol
fractionated by HPLC, column: MultoHigh RP18 5 µM (125x2.0 mm column); gradient: 0 min: 50% water, 50% methanol

5 min: 50% water, 50% methanol

25 min: 10% water, 90% methanol

25.1 min: 100% methanol

SecinH3 elutes at ca. 20 min; analyzed by MS

We thank Klaus Rotscheidt for help in establishing this protocol.

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Extraction of the SecinH3 from mouse liver

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Acknowledgements

Associated Publications

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