MarkerGene™ in vivo lacZ β-Galactosidase Intracellular Detection Kit

Product ID: M0259



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

Availability: In stock


Description

This kit allows ultra-sensitive detection of β-galactosidase activity in live mammalian, yeast, bacterial, or plant cells. It is especially useful for flow cytometry applications.

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 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.

Although chromogenic assays of beta-galactosidase activity (i.e. X-Gal) have use, the recent application of the fluorogenic substrate fluorescein di-β-D-galactopyranoside (FDG) combined with fluorescence microscopic analysis (confocal microscopy) analysis has been shown to be several orders of magnitude more sensitive. In addition, because of its improved detection limits, the FDG substrate has found extensive use in automated ELISA type assay systems .

This kit uses the beta-galactoside analog fluorescein di-β-D-Galactopyranoside (FDG) in a protocol that sensitively distinguishes lacZ+ vs. lacZ- cells. See also Product M0250 for more information and references.


Technical Data
SKU M0259
Unit Size 1kit
Detection Method Fluorescence


References and Citations

Citations:

  • Zhulyn O, Nieuwenhuis E, Liu YC, Angers S, Hui CC. (2015) "Ptch2 shares overlapping functions with Ptch1 in Smo regulation and limb development." Dev Biol. 397(2):191-202.
  • Akita H, Ito R, Kamiya H, Kogure K, Harashima H. (2007) "Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid-mediated transgene expression." J Gene Med. 9(3):197-207.
  • Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, Gustafsson E, Chandraker A, Yuan X, Pu WT, Roberts AB, Neilson EG, Sayegh MH, Izumo S, Kalluri R. (2007) "Endothelial-to-mesenchymal transition contributes to cardiac fibrosis." Nature Medicine 13: 952-961.
  • Akita H, Ito R, Kamiya H, Kogure K, Harashima H. (2007) "Cell cycle dependent transcription, a determinant factor of heterogeneity in cationic lipid-mediated transgene expression." The Journal of Gene Medicine, 9(3): 197-207.
  • Kawashita Y, Ohtsuru A, Fujioka H, Kamohara Y, Kawazoe Y, Sugiyama N, Eguchi S, Kuroda H, Furui J, Yamashita S, Kaneda Y, Kanematsu T. (2000) "Safe and efficient gene transfer into porcine hepatocytes using Sendai virus-cationic liposomes for bioartificial liver support." Artif Organs. 24(12):932-8.
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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 GP. (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: Can this kit be used to stain fixed cells and tissues?
The following protocol can be used for staining fixed cells with the M0259 kit:
  1. Cells should be washed 3X with sterile 1X PBS
  2. Add an appropriate volume of fixant (1.85% Formaldehyde/ 0.35% Glutaraldehyde)
  3. Incubate at room temperature for 30mins
  4. Remove fixant and wash cells 3X with 1X PBS
  5. Prepare 500uM Substrate reagent in PBS immediately prior to use and apply to cells
  6. Incubate at 37C for 30mins to 2 hours
  7. Analyze as required

Analysis should be carried out as soon as possible as fixing the cells will cause the dye to leak out of the cells.

For staining of tissue sections we recommend cryostat sections that have not been fixed, as fixation will lead to very diffuse staining. Tissue should be soaked in a solution of the substrate reagent at a concentration of 0.5 to 2 mM for 30 minutes to 2 hours at 37C. Tissue is then washed prior to observation. Keeping the Tissue cold will help prevent translocation of the fluorescent signal.

⚛ Question: I am getting high amounts of background/endogenous staining. How can I prevent this? When I use the PETG solution the cells die.

It is not unusual to see some background staining with this method, since the substrate in this kit, FDG, is quite labile. The obvious problems are from hydrolysis of the substrate prior to staining. If the staining solution is already quite yellow prior to staining, it may be already hydrolyzed and not usable. There is always a slight tan color, especially for the concentrated stock in the kit, but a bright yellow color indicates decomposition.

Our QC prior to shipment requires a background fluorescence reading of

There is a slight chance that there is just endogenous b-Gal activity (usually lysosomal activity) that is responsible for the high background. We include in the kit the inhibitor, chloroquine, which is a specific inhibitor of this activity. You might try that protocol. Additional inhibitors for this type of assay include verapamil and probenicid. Please see this article for more information about these techniques.

The IPTG is an broad based inhibitor and is not really useful for live cell analysis. It is useful for kinetic assays to "stop" the enzyme completely.

⚛ Question: How do I use this kit to stain yeast cells?
The following protocol may be used for staining yeast after growing the cells:
    1. Pellet the yeast cells by centrifugation.
    2. Remove growth medium without disturbing pellet.
    3. Resuspend pellet in 1 ml of 70% ice cold ethanol.
    4. Shake vigorously for 5 minutes on ice.
    5. Centrifuge the cells again.
    6. Remove 70% ethanol without disturbing cell pellet.
    7. Resuspend the pellet in ice-cold 100 ul Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 0.27% mercaptoethanol. The mercaptoethanol should be added right before use. Z buffer without mercaptoethanol should be stored at 4C).
    8. Immediately add 100 ul of ice-cold 100 uM FDG in 100 ul Z buffer. The FDG may be prepared by diluting 10 ul of the 10 mM stock FDG substrate provided with the kit into 990 ul of Z buffer.
    9. Incubate for 20-30 minutes at 37C.
    10. Measure fluorescence by flow cytometry or microscopy.

The reaction may be stopped via the addition of Na2CO3 (142 mM final concentration), otherwise the reactions will require careful timing in order to ensure all reactions are analyzed at the same time after addition of the FDG substrate.

Please note Z buffer and 70% ethanol are not included in this kit.

⚛ Question: We recently ordered the "in vivo LacZ beta-galactosidase detection kit", which was delivered today. Unfortunately, the Kit was already defrosted. Do you have any information, if this is harmful for the reagents?

The kit should be fine, if left out for a few hours at room temperature. Certainly store it in the freezer (-20oC) upon receipt until use.

The only potentially temperature-sensitive sample in the kit is the fluorescent substrate reagent. Evidence of decomposition will be evident by a change in color of the fluorescent substrate reagent solution to a bright yellow (fluorescent) color and a noticeably high background level of fluorescence for the "blank" samples. Please note: this solution normally has a tan or light yellow color because it is so concentrated.

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