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______________________________ Reply Separator _________________________________
Subject: Re: lacZ detection
Author: dbk@aber.ac.uk at INTERNET
Date: 04/03/96 06:20
> From: DR M AL-RUBEAI <ALRUBEAM@novell1.academic-computing-service.bi
rmingham.ac.uk>
> Organization: The University of Birmingham
> To: Cytometry Mailing List <[7f]>
> Date: Thu, 29 Feb 1996 09:03:58 BST
> Subject: Re: lacZ detection
> Reply-to: M.Al-Rubeai@birmingham.ac.uk
> Priority: normal
> 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 concentrati
on
> 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.
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*Edward Llwyd Building Fax: +44 1970 622354 *
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*University of Wales, Email: dbk@aber.ac.uk *
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