This point is certainly important, and well taken. It may well be the major cause of the problems of irreproducibility to which Joerg refers. Also NB that even after any bulk gradients have been collapsed by val/nig (WHICH DON'T ALWAYS WORK PROPERLY IN GRAM NEGATIVE BACTERIA NOR STATIONARY PHASE YEAST, INCIDENTALLY, NOR IN THYLAKOIDS, which is another story) there will still be a DONNAN potential because there are a lot of internal 'fixed' charges (mainly proteins, RNA) which cannot cross the cell wall, whatever shape the cytoplasmic membrane is in, even if it has been dissolved completely in fact. The DNA microarray studies, for instance, from DeRisi et al onwards (DeRisi, J. L., Iyer, V. R. & Brown, P. O. (1997). Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680-686), tell us that these components changes rapidly and substantially - and of course, as this group wlel knows, that statement applies to BULK, macroscopic measurements, and doesn't take into account the inter-cellular heterogeneity reveled by flow and image cytometry. The Donnan potential will be exactly balanced by a Donnan pH gradient of opposite sign, and that intenral pH (and the pH gradient if the outside pH is clamped by a high buffer concn - and it needs to be high) will thus depend on the amount of RNA and proteins being expressed in that cell. Oh happy days..... All the best, Douglas. > We haven't ever done pH in microorganisms but have done so in human > blood cells - platelets, granulocytes, now doing it in monocytes, > and in phagocytic compartments (all are published). In all cases we > used BCECF and calibrated using nigericin. However, that has to be > done in a buffer system whose K+ concentration mimics that of the > interior of the cell, because nigericin collapses the ratio of > H+in/H+out to equal that of K+in/K+out, which therefore needs to > equal one. That seems to be the thing people who've contacted me in > the past don't take into consideration. If one takes that > precaution, we have had very, very reproducible calibration curves. > Elizabeth R. Simons > > joerg ueckert wrote: > > > > Folks, > > > > We're holding us busy by applying CFDASE for ratiometric determination of > > internal pH in bacteria and yeast. Apart from strain- and pre-history - > > dependent difficulties in dye uptake the generation of a 'useful' calibration > > curve is the biggest challenge. The well-known combination > > valinomycin/nigericin results in a high degree of variability and curve shifts > > upon repetition. Also, apart from these, other agents (e.g. alcohols) or > > methods (e.g. electroporation) to facilitate equilibration of internal and > > external pH give stunning differences compared to mentioned 'standard' method. > > All remarks, suggestions and experiences with agents which provide reliable > > calibration curves for pHi in microorganisms are welcome. Providing a > > sufficient number of responses, I will post a summary on the list. Thanks in > > advance. > > > > - Joerg > > > > ------------------------------------------------ > > Joerg Ueckert > > Unilever Research > > Microbiology > > Vlaardingen, NL > > > > Email joerg.ueckert@unilever.com > > ------------------------------------------------ > > -- > Elizabeth R. Simons, Ph.D. > Professor of Biochemistry > Boston University School of Medicine > 715 Albany Street, K 602 > Boston, MA 02118 > phone (617) 638-4332 > FAX (617) 638-5339 > email: esimons@bu.edu > (Prof.) Douglas B. Kell, Cledwyn Building, Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DD Tel: +44 1970 622334 Fax: +44 1970 622354 dbk@aber.ac.uk http://gepasi.dbs.aber.ac.uk/home.htm
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