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So the paper I am reading (here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094582/) uses densitometric units to quantify the western blots.

The authors mentioned that the unphosphorylated proteins were used as loading controls, and their quantities are represented by the densitometric graphs for each corresponding figure, but protein activity is measured by their phosphorylated counterparts which I believe is represented by the densitometric graph on the right of each corresponding figure.

Questions:

  1. Did I interpret that correctly?
  2. What exactly are loading controls?
  3. I am trying to use these data for parameter estimation purposes. Is it possible to find the basal mass quantity of a particular protein?
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  • $\begingroup$ There are several questions here, note that we usually ask for a single focused question. $\endgroup$ Nov 12 '20 at 20:32
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Answers to each question:

  1. Approximately, yes. In this case, phosphorylated Akt (p-Akt) is supposed to be the active form of the enzyme that goes around the cell doing its job, whereas non-phosphorylated Akt doesn't have activity that they are interested in. The authors do show that targets of Akt show characteristics of Akt activity when Akt is phosphorylated (Fig 2B, 2C).

Note that densitometry is just a way of measuring the intensity of some bands on immunoblots (westerns). In this case, it is normalized by the loading control (non-phospho Akt). So you could interpret the densitometry plots as showing the proportion of Akt that is phosphorylated. It is a little confusing because they subsequently normalize them to a specific timepoint. From the methods:

The results were quantitated by densitometric analysis (ImageQuant, Molecular Dynamics and PDSI, GE Healthcare). The ratio of phosphoprotein to its respective internal control was normalized to the control level at 0.5 h, arbitrarily set to 1.

  1. Loading controls indicate how much protein of interest is present. For example, 1% of 100mg of protein is the same as 10% of 10mg protein (each is 1 mg). However the interpretation of 1% vs. 10% may be quite large. So if what you are interested in is a percentage or proportion, you need to have a loading control to allow unbiased comparison or normalization.

  2. I don't really know what "basal mass quantity" means, but you could look into the molecular weight of proteins (usually measured in kilodaltons). For example the molecular weight of Akt is ~55kda. You can compute this from the protein sequence by adding up the weights of all the amino acids.

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