Extraction of the SecinH3 from mouse liver
Lab/Group: Famulok Lab
Related Journal & Article Information
Journal: Nature
Introduction
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.
Materials
Reagents
ca. 300 mg mouse liver
Chloroform
Methanol
PBS
Equipment
HPLC/MS: Agilent 1100 Series HPLC with Bruker Daltonics Esquire Series MS
HPLC columns:
1. 10×4.0 mm with Nucleosil RP18 5 µm
2. 125×2.0 mm with MultoHigh RP18 5 µm
Polytron (Kinematica)
SpeedVac
Procedure
1. 300 mg mouse liver each weighed into 15 mL centrifugation tubes and taken up in 1 ml PBS, 2 mL chloroform added
2. mouse liver homogenized by polytron
3. incubated at RT for 30’ under agitation,
4. Centrifuged 5’ at maximum speed
5. Chloroform phase transferred into fresh centrifugation tube
6. Water phase extracted second time on shaker with 1.5 mL chloroform, 20’
7. Centrifuged 5’ at maximum speed
8. Chloroform phases combined and solvent removed on SpeedVac
9. Residue dissolved in 100 µL/(100 mg liver) methanol, centrifuged 5’ at 14.000 rpm and liquid transferred into HPLC glass vial
10. Fractionated via HPLC on Nucleosil RP18 material 5 µm (10×4.0 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
11. solvent of collected fractions removed by SpeedVac and residue taken up in 50 µl methanol
12. fractionated by HPLC, column: MultoHigh RP18 5 µM (125×2.0 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
13. SecinH3 elutes at ca. 20 min; analyzed by MS
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
We thank Klaus Rotscheidt for help in establishing this protocol.
Keywords
SecinH3,
mouse liver
high performance liquid chromatography (HPLC)
Mass spectrometry