Hani Jouihan Author Answered Mar 27, 2013
Division of Endocrinology, Gerontology and Metabolism, Stanford University School of Medicine, USA
1) our spectrophotometer does not have a 540nm filter. We do have 535 and the tech seemed to think that this was "close enough". any thoughts on that?
? This is good enough.
2) what is your procedure for creating the glycerol standard titrations to generate the regression line from the data?
? This is to be determined empirically. You might want to look up some of the available commercial kits to see what concentrations of standard they have use. I would usually have a bulk idea on the expected TG amounts, so I will tailored my standard concentrations to for my use.
3) is there a way to add steps to obtain a protein fraction from this protocol?
? You can not use the same piece of liver to get TG and protein. The chemical treatment used to extract TG will not be suitable to do protein determination. Alternatively, you can dedicate another piece from the same tissue to do protein analysis.
4) we are planning to use the protocol on both muscle and triglyceride (which the literature says is fine). have you had success with both?
? What do you mean by “Muscle and TG”? I think you meant muscle and liver. In any case, this procedure should work on any tissue that you suspect to store TG.
5) we may not have 100mg of tissue for all samples. if we have 50mg, for example, must we adjust the volume of all the reagents or does this not matter b/c the calculations are based on total weight of tissue?
? This you have to adjust the volumes and calculations. I did not have this issue because liver and muscle usually provide ample material. If you are dealing with a mouse model that store excessive amounts of TG, you can use less tissue material.