Talk:MiPNet15.08 TPP electrode
Pmt, PC from literature?
Question: (1) With the TPP spreadsheets, Pmt and Pc values are the values that we use literature values for (that actually measured membrane content), and not actually measured protein (mg/mL) off the sample that is being run? and Vmt I think we also used published values for?
Answer: No, Pmt and Pc describe how much sample you put into your chamber and therefore can not be taken from literature. If you use twice the amount of sample these values will have to double. For isolated mitochondria Pmt = Pc. Please see also the Rottenberg paper cited.
Vmt can be taken from literature, especially as the TPP method is very insensitive against errors in Vmt. Similarly we take Kin and Kout from literature. Vmt, Kin , and Kout are all SPECIFIC values, that is values per amount of mitochondrial / cellular protein. But to use these specific values you need the absolute values of mitochondrial cellular protein- ideally from an assay or at least some rough estimation from the respiration itself (if you know the typical respiration of your sample per mg mitochondrial protein). In the sheet "external input" you see a very rough template for this respiration approach, but a proper assay is of course the way to go.
Some background information :-) : Actually the choice of mitochondrial protein as the marker the specific values Kin and Kout is a bit arbitrary / counter intuitive: The unspecific binding, described by Kin and Kout, actually will take place mainly in the mitochondrial lipids not in the proteins. But since mitochondrial protein is easy to measure and the ratio lipid to protein is hopefully constant all the initial developers of the method used mitochondrial protein as a marker for the amount of mitochondria. For samples beyond isolated mitochondria (permeabilized cells,..) the assumption of a constant lipid to protein ration of course brakes down, maybe one reason why there are no good values for Kout in "cellular samples". Fasching Mario 09:41, 17 April 2015 (CEST)
