lacZ detection

Douglas B. Kell dbk at aber.ac.uk
Fri Mar 1 12:36:56 EST 1996


> From:          DR M AL-RUBEAI <ALRUBEAM at novell1.academic-computing-service.birmingham.ac.uk>
> Organization:  The University of Birmingham
> To:            Cytometry Mailing List <>
> Date:          Thu, 29 Feb 1996 09:03:58 BST
> Subject:       Re: lacZ detection
> Reply-to:      M.Al-Rubeai at 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 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.


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