Ultrasensitive β-Galactosidase substrate for live cell and tissue (in vivo and in vitro) assays. See references below for numerous applications of this widely used substrate.
NOTE: Molecular Biology grade: <20 ppm background fluorescence QC specification.
Absorption <300nm; upon enzymatic cleavage 490nm.
Emission none; upon enzymatic cleavage at pH 9 514nm.
|Solubility:||Soluble in H2O, EtOH, DMF or DMSO.|
|Background / Technical Information:||NOTE: The following information is given as a viable methodology for use of the β-Galactosidase Sample Kit for in vitro and in vivo lacZ β-Galactosidase detection. The user may determine their own best conditions for use dependent on the specific conditions present in their experiment. This information is subject to change without notice.|
One of the most common reporter genes used in molecular biology application is the E. coli lacZ gene that codes for an active subunit of β-Galactosidase in vivo. Since this enzyme is generally absent in normal mammalian, yeast, some bacterial and even plant cells; it can be detected at very low levels. Also, since the enzyme has a wide substrate specificity, monitoring lacZ expression (and therefore co-expressed genes or promoter efficiency) has become routine to the point of detection of as few as 5 copies of β-Galactosidase per cell by FACS analysis.
Although chromogenic assays of β-Galactosidase activity (i.e. X-Gal) have significant use, the recent application of the fluorogenic substrate have been shown to be several orders of magnitude more sensitive.
A.) Fluorescent Substrate Reagent. Make a 10 mM solution of the fluorescent substrate Fluorescein di-β-D-Galactopyranoside (FDG) by dissolving FDG first in a small volume of organic solvent (EtOH, DMF, DMSO) and diluting with H2O or buffer. Note: to avoid decomposition, cool solutions in ice-water prior to mixing. Do not heat or ultrasonicate FDG solutions as this may also cause unwanted decomposition. See also Note C. below.
B.) Reference Standard. It is recommended that a reference standard be prepared (2 mM Fluorescein in distilled water) for quantitating results.
C.) Storage and Handling. Fluorescent reagents and fluorescent labeling solutions or samples should be handled with care, kept cold (ice bath) when not in use, and stored frozen (-20°C). In case of contact with skin or eyes, wash thoroughly with soap and water. Reagents should be stable for at least 6 months following purchase. Unstable background fluorescence readings for blank samples will indicate decomposition. These materials are intended for research purposes only. Please contact us for information on use or licensing.
III. ASSAY CONDITIONS
It is recommended that measurements be made in duplicate, if possible, and hat the approximate concentration range of the fluorescent probe be adjusted for optimum signal and sensitivity. Previous studies have indicated that the labeling of cells is virtually independent of the initial fluorescent probe concentration in the range of about 100 µM 2mM. Since staining may be somewhat time dependant, a time course for the experiments should also be generated for initial trials. The emission of the highly fluorescent product fluorescein is monitored at 512 nm using excitation at 488 nm (argon ion laser). The user is asked to consult with the manufacturer (or instrument manual) for the particular instrument in use for the appropriate filter set(s) needed for monitoring at these wavelengths. Typical epifluorescence microscopic analysis is performed using an excitation filter, a dichroic filter and emission filter for Fluorescein fluorescence. For flow cytometric analysis, the FACS instrument is typically equipped with bandpass filter for monitoring fluorescein fluorescence.
The working concentration range of the assay will need to be determined for each individual experiment. A blank prepared with all reagents, substituting corresponding lacZ negative cells should be run in parallel if possible. Alternately, a blank prepared with all reagents except cells (substituting water of media) can be run to determine background fluorescence readings for each experiment.
Product Literature References
A microplate fluorimetric assay for transfection of the beta-galactosidase reporter gene
: V.A. Rakhmanova & R.C. MacDonald; Anal. Biochem. 257
, 234 (1998), Abstract
Fluorescent probes for living cells
: I. Johnson; Histochem. J. 30
, 123 (1998), Abstract
General Literature References
Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ
: G.P. Nolan, et al.; PNAS 85
, 2603 (1988), Abstract
Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: an assay for serum ferritin
: R. Armenta, et al.; Anal. Biochem. 146
, 211 (1985), Abstract
The homogeneous substrate-labeled fluorescent immunoassay
: J.F. Burd; Meth. Enzymol. 74 Pt C
, 79 (1981), Abstract