Measuring intracellular protein concentration: why and how?

In discussions of cell volume regulation, an assumption is often made that a cell can be characterized by its “natural” volume determined by some intrinsic set point. Although this may be true for stable and differentiated tissues, it is obviously wrong for actively dividing cell populations, where the volume doubles between divisions. In this case, it seems more natural to talk about a set point for protein concentration C, rather than for volume V. For example, the majority of data on regulatory volume increase or decrease have been collected within a brief period following the osmolarity change, presumably to avoid complications resulting from cell growth. By using the intensive property of protein/water concentration, rather than the extensive property of volume, this complication can be avoided. Another example comes from apoptosis. Apoptotic shrinkage can be caused by dehydration or by fragmentation. To distinguish between these two processes, one has to know both V and C. Thus, we have developed two simple methods based on light transmission microscopy, to measure these parameters. Cell volume is measured by adding a strongly absorbing extracellular dye to the medium. If a transmission image is taken at a wavelength of maximal dye absorbance, cells appear brighter than the background in direct quantitative relationship to their thickness. This method gives accurate information on V and has been used extensively in our lab. At the same time, one can collect brightfield images away from the absorbance peak. There is a theory that relates transmission image intensity to the phase delay by means of the transport‐of‐intensity equation (TIE). We have further improved the accuracy of TIE by introducing additional calibration, so that protein concentration can now be expressed as C(g/ml) = 1.0·∑ϕ(radians·mm 2 )/V(mm 3 ), where ϕ is the output of TIE and 1.0 is an empirical coefficient. This approach gives a new insight into cell adaptation to anisosmotic environment and into apoptotic shrinkage.