I am interested in measuring the composition of some organelles (percentage of Lipids, proteins and carbohydrates). Please suggest some good methods. if possible, please add some references
PGC1-alpha is the indicator of the biogenesis of mitochondria, so that WB analysis of PGC1-alpha is useful to understand the tendency of the “mitochondria-genesis”. Further, PPAR-gamma is the nuclear transcription factor and the master regulator for the TCA cycle and lipid metabolism which mainly occurs in the mitochondria.
Staining Strategy
Why not use MitoSOX™ Red, the fluorescent chemical which reacts to the electron transport chain in the inner membrane of mitochondria? This mitochondrial superoxide indicator is useful for live-cell imaging and the fluorescence is maintained even after you have fixed and stain with mitochondria-related antibody such as PPAR-gamma or PGC1-alpha.
If you're interested in mitochondrial degradation, you can evaluate the levels of different mitochondrial proteins (TOM20, TIM23, etc.) using immunocytochemistry and western blot analysis. Purification of mitochondria before WB is highly recommended.
You can also separate the attached fraction from the inserted fraction of mitochondrial proteins using a biochemical alkali extraction method. Here is the link for the paper:
Thanks for your time and suggestions. Actually i am looking how to measure the percentage composition of carbohydrates, proteins and lipids in an organelle. We want to isolate e.g mitochondria and determine its composition/constituents like proteins, lipids, carbohydrates nucleic acids by mass. I am not interested in organelle turnover or degradation.
I am sending you two papers which showed how to isolate and characterized the contents (chemical analysis of protein, carbohydrates, amino acids and lipids) in the organelle.