Thank you very much for the protocol. I have couple questions...

JH
Jessica Han
Mar 6, 2014
protocol Protocol: Measurement of Liver Triglyceride Content
Thank you very much for the protocol. I have couple questions need help with
1) How much supernatant do you expect at step 12? We use the protocol for high fat mice liver sample, and only got 30-50 ul supernatant. Is it normal? Should we digest longer at the steps 3-6?

2)C
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Jessica Han Answered Mar 7, 2014

North Carolina A&T State University

At step 9, after bring up to 1200 ul with 1:1 H2O:Ethanol, can this solution be stored at -20 for later process?

Thanks again.

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Jessica Han Answered Mar 7, 2014

North Carolina A&T State University

Thanks very much! I will do different extraction volume of ethanolic KOH. Another question is what is the centrifuge speed at step 8 and 12?
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Hani Jouihan Author Answered Mar 7, 2014

Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA

Hi Jessica
I can't recall exactly how much sup to expect from step 12. However, since you are using a HF fed mice, you might want to use couple of different volumes of ethanolic KOH, and while you at it, try to incubate longer. The more effort you put upfront in optimizing the assay, the better off you will be in the long term.
Yes, sups can be stored at -20C. If you anticipate repeated measurements of the same sample, prepare aliquots to prevent freez-thaw issues.
Hope this helps
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