Abstract
This assay is designed to measure relative lipid accumulation of experimental treatments compared to controls. The reagent measures the concentration of glycerol released after lysing the cells and hydrolyzing the triglyceride molecules. The triglyceride concentration can then be determined from the glycerol values.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was previously used in Norris et al. (2003).
References
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You can check papers from Dr. Ronald Kahn @ Harvard, you should be able to see this protocol referenced in one of his older papers. Alternatively, your PI can pay $$$ to buy commercially available kits.Don't know if assay will work with the other conditions mentioned.Best
Hi JessicaI 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
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?
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.
Hi Bronsonthe purpose of using MgCl2 is to serve as a precipitating agent for lipids. For more details, I recommend googling for "Triglyceride Assay and magnesium chloride" you will find a lot of detailed info about this topic.
Hi BronsonA short answer is; No, I have not used serum TG kit to measure the liver TG. However, since TG should be the same regardless of the origin, I won't be surprised to see that you can use the serum kit with liver sample. The only trick here would be the way you are going to extract the TG from liver. So, if you decide to process the liver to extract the TG, make a serial dilution of this extract and test them all at once. This is important since liver might have way more TG than normally found in serum and the kit dynamic detection range might not detect very hi TG levels. Therefore, by diluting your extracts (1:2, 1:5, 1:10...ect) you might get it to work.Good luck
Hi WilI would assume it should work, especially that you have already separated the plasma, so no blood-cells would interfere with the reading. However, I never tested plasma. If you decided to try the protocol with plasma, make sure to include a positive control (from liver, for example) and used several different volumes of plasma so that your values are withing the linear range of the standard curve.Good luck
Park, seeing the data sheets does NOT mean that the reagents are commercially available. If you happen to still have reagent A, use it. If not, then you get the alternative, which happens to be the "Free Glycerol Reagent (F6428)". Check the dictionary to see what "replace" meansI did not mention that you need to mix TG reagent A and Free glycerol reagent
Hi ParkSince I did not use Sigma's new replacement product, I can't precisely tell you what to use.However, you can optimize the assay according to your needs by keeping the ratio of sample: reagents at a constant. this way, you can reduce the total assay volume (let's say from 1000ul to 200 ul) by dividing all reagents and sample volumes by 5. this will keep the actual ratios among reactants constant, despite the reduction in assay total volume. Like any newly introduce change in experiments, it is advisable that you test few different times before you reach a reproducible result. As long as you always have your standard included in each run, you should be OK in terms of extrapolating your experimental values.
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.
There is no universal equation for calculating TG concentration. You can check protocols of commercially available TG kits and you will see several methods/equation for calculation. In the equation, the (10/30) is the ratio of standard used (10ul) to the total volume of test sample which is 30ul. The (415/200): the 41corresponds to the volume (415ul) from where the tested sample came out (step 10: Move 200 µl to new epp tube, add 215 µl 1M MgCl2, vortex)The 200 is for the 200ul that we took out of step 9Finally the 0.012 corresponds to the the final volume obtained in step 9 (1200ul) Hope this helps
