MarkerGene™ FACS Fluorescent Blue lacZ β-Galactosidase Detection Kit

Product ID: M0255



Unit SizePriceQuantity 
1kit
$257.92
  • Buy 5 for $206.34 each and save 20%

Availability: In stock


Description

This flow cytometry kit can be used in conjunction with eGFP or RFP assays since it allows highly sensitive detection of β-galactosidase activity in mammalian, yeast, bacterial, or plant cells, at a shorter wavelength (blue fluorescence) than other methods. It is especially amenable to co-staining with a green (fluorescein, GFP) labeling for dual labeling experiments.

One of the most common reporter genes used in molecular biology applications is the E.Coli lacZ gene that codes for an active subunit of beta-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, and 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 beta-galactosidase per cell by FACS analysis.

Although chromogenic assays of beta-galactosidase activity (i.e. X-Gal) have use, application of the fluorogenic substrate 3-carboxyumbelliferyl β-D-galactopyranoside (CUG) combined with Fluorescence Activated Cell Sorting (FACS) analysis has been shown to be several orders of magnitude more sensitive. In addition, because of its high water solubility and detection limits, the CUG substrate has found extensive use in automated ELISA type assay systems. This assay is especially useful when co-staining with a fluorescein-type cell label (FITC-antibody, fluorescein based substrate, etc.) for dual wavelength detection. Many new instruments have multi-color FACS detection capabilities (see the BD LSRII system for example), that can utilize this new kit. Please consult your instrument manufacturer brochure for more information.

The FACS Blue lacZ β-Galactosidase Detection kit provides all the reagents and a detailed protocol to perform up to 500 automated (5 x 96 well microtiterplate) assays.


Technical Data
SKU M0255
Unit Size 1kit
Detection Method Fluorescence

References and Citations

Citations:

  • Jan TA, Jansson L, Atkinson PJ, Wang T, Cheng AG (2016) Profiling Specific Inner Ear Cell Types Using Cell Sorting Techniques. ed Sokolowski B (Springer New York, New York, NY), p 431.
  • Jan TA, Chai R, Sayyid ZN, Cheng AG. (2011) "Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry." J. Vis. Exp. (58) e3432.
  • Kalani MYS, Cheshier SH, Cord BJ, Bababeygy SR, Vogel H, Weissman IL, Palmer TD, Nusse R. (2008) “Wnt-mediated self-renewal of neural stem/progenitor cells.” Proc Natl Acad Sci U S A. 105(44): 16970–16975.
  • Heidkamp MC, Scully BT, Vijayan K, Engman SJ, Szotek EL, Samarel AM. (2005) "PYK2 regulates SERCA2 gene expression in neonatal rat ventricular myocytes." Am J Physiol Cell Physiol. 289(2):C471-82.
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References:

  • Roederer M, Fiering S, Herzenberg LA. (1991) "FACS-Gal: Flow Cytometric Analysis and Sorting of Cells Expressing Reporter Gene Constructs." Methods: Companion to Meth. Enzymol.2: 248.
  • Nolan GP, Fiering S, Nicolas JF, Herzenberg LA. (1988) "Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ." Proc. Natl. Acad. Sci. U S A 85: 2603-2607.
  • Krasnow MA, Cumberledge S, Manning G, Herzenberg LA, Nolan G.P. (1991) "Whole animal cell sorting of Drosophila embryos." Science 251: 81-85.
  • Fiering S, Nolan G, Herzenberg LA. (1988) "Inhibitors of endogenous mammalian beta-galactosidase (B-Gal) or transduced E. coli B-Gal improve the sensitivity and versatility of the FACS-FDG fluorogenic assay for E. coli lacZ expression in viable mammalian cells." Cytometry (Suppl 2) 1.
  • Guo W, Wu H. (2008) "Detection of LacZ expression by FACS-Gal analysis" Nature Protocols DOI: 10.1038/nprot.2008.163 http://www.natureprotocols.com/2008/08/06/detection_of_lacz_expression_b.php.
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Technical Support

Question about this product? Ask a Scientist!

We pride ourselves on the high quality of our products and want you to get the best possible results from your assays. If you have any questions about this product or need help optimizing your protocol check out the product FAQs below or ask your own question and one of our expert scientists will get back to you asap:


⚛ Question: Could this kit be used to measure the SA-beta-Gal activity of human senescent cells?
This kit functions at the wrong pH for SA-beta-Gal. The recommended kit for measuring SA-beta-Gal is M1405 (MarkerGene™ Fluorescent Cellular Senescence Microtiterplate Assay Kit).
⚛ Question: What is the absorbance of the substrate?
The absorbance of the substrate is 330nm.
⚛ Question: For FACS do the cells need to be incubated with the reaction buffer or permeabilized with Triton-x 100?
The reaction buffer is not used for FACS, the cells are incubated with appropriate medium containing the substrate and there is no need for permeabilization.
⚛ Question: How do I use the standard reagent?
The reference standard solution included in the kit is for use when using a plate reader for: a)calibrating your reader, such as gain setting, filter setting etc. b)determine the linear range of coumarin, which is the product of the substrate. All assays should generate product which is in the linear detection range by fluorometer or fluorescence plate reader.
⚛ Question: Is the substrate in this kit compatible with a violet (405nm) laser?
The optimum excitation wavelength is 386 nm. Excitation at 405 nm, although not at the absorption maximum for the dye, should still work fine for the assay. The absorption spectra for these coumarin dyes is typically quite broad in nature. In acidic conditions (below pKa 7.8) the absorption shifts to lower wavelengths (235-340 nm) and fluorescence intensity decreases.
⚛ Question: Is it possible to put the CU labeled cells in culture, or is the labeling reaction lethal?
The CUG and the carboxyumbelliferone (fluorescent product) are not toxic, and you should be able to re-culture your cells after sorting. Keep in mind that some cell toxicity may occur just because of the many manipulations in the FACS sorting, but the staining should not affect viability more than another other type of staining protocol.
⚛ Question: How long will the Substrate Reagent remain stable?
CUG is more stable than FDG for these assays. Still, some care should be exercised when using the kit. The CUG substrate should be stable in solution for at least several hours and even several days, if kept cold. Acidic or basic buffers will hasten the decomposition. Evidence of decomposition can be monitored by running a blank (non-lacZ cells) at the beginning and end of your assays.