 |
 |
 |
 |
 |
> John
>
> The best flow cytometry substrate for the detection of lacZ activity
> is a lipophillic FDG derivatves with 2, 4, 8 and 16 carbon acyls
> known as ImaGene (Molecular Probes). However, this substrate has a
> problem for the quantification of lacZ activity. We have used it to
> assay b-gal activity in insect cells infected with baculovirus. A concentration
> equilibrium between cell and supernatant of the hydrolysed substrate,
> irrespective of enzyme activity, meant that lacZ+/lacZ- resolution
> was impossible. Quantitative measurements proved problematic because the
> enzyme activity showed no sign of saturation by the substrate.
> Good luck
> Mohamed Al-Rubeai, Ph.D.
> School of Chemical Engineering
> University of Birmingham
>
> es, let me agree. These probes are just not what they are cracked
> up to be. We wasted some important time and scarce money on this
> type of stuff in bacteria - only bugs that are in absolutely
> tip-top condition keep the product in. Most bugs are more than
> slightly leaky (and actually dormant ones of a species which is an
> obligate respirer can be completely leaky, and can reseal as part
> of resuscitation, which if you ever believed in the chemiosmotic
> coupling hypothesis might cause you to cease so doing,but that is
> another story (Votyakova, T. V., Kaprelyants, A. S. & Kell, D. B.
> (1994). Influence of viable cells on the resuscitation of dormant
> cells in Micrococcus luteus cultures held in extended stationary
> phase. The population effect. Appl. Env. Microbiol. 60,
3284-3291.))
Bottom line: most of these things don't work if you want to be
quantitative.
Even more bottom line to manufacturers: but we'd like them to.
********************************************************************
*Douglas B. Kell Phone: +44 1970 622334 *
*Edward Llwyd Building Fax: +44 1970 622354 *
*Institute of Biological Sciences *
*University of Wales, Email: dbk@aber.ac.uk *
*Aberystwyth SY23 3DA, U.K. http://gepasi.dbs.aber.ac.uk/home.htm *
********************************************************************
********************************************************************
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web