Protocol

TD-P Revision 1. Creation Date: 8/2/2018

Revision Date: 2/20/2020

Gus Gene Assay in Transformed Tissues

Protocol by Dr. Paul J. Bottino (retired), Plant Molecular Genetics, University of Maryland

Introduction
Gene reporter systems have become an invaluable tool for the study of gene expression
regulation in plant research. In these systems, a gene reporter, usually an enzyme, is fused to a
specific gene promoter, leading to transcription of the gene reporter under control of the
promoter. Then, the enzyme activity can be measured and used as indication of gene
expression levels. Of the many reporters in use today, β-glucuronidase (GUS) is the most
popular and has been particularly useful in helping identify transgenic events in plants due in
part to its stability in various conditions and use in various sensitive assays. E. coli
βglucuronidase catalyzes the cleavage of various β-glucoronides, has a molecular weight of 68.2
kDa, and appears to function as a tetramer. It is very stable in a variety of conditions and is
most active in the presence of thiol reducing agents such as β-mercaptoethanol (βME) or DTT.
In addition, it may be assayed at any physiological pH and it has optimal activity between pH 5.2
and 8.0. The original GUS reporter gene reporter was developed as a gene fusion marker in E.
coli and in C. elegans. However, currently it is used extensively to monitor chimeric gene
expression in plants. Interestingly, there is little or no detectable β-glucuronidase activity of
yeast, Drosophila, C. elegans, Dictyostelium, or in almost any higher plant. In Agrobacterium,
however, some of the GUS plasmids showed significant GUS activity even in the absence of a
promoter. Thus, in order to use this system in Agrobacterium-mediated transformations and
accurately measure gene expression regulation by specific promoters, one laboratory
constructed GUS genes carrying an intron, which must be processed before expression takes
place. Thus, allowing the monitoring of Agrobacterium-mediated transfer and expression of
foreign genes in plants. Here, we describe how to use the GUS system in a histochemical assay
to perform a qualitative analysis of β-glucuronidase activity in tissues and cells from
transformed organisms. In addition, we describe a fluorimetric assay, which allows us to
quantify β-glucuronidase activity in tissues and cells transformed using the GUS reporter gene
system.

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Gold Biotechnology / FM-000008 TD-P Revision 1.0
GUS Gene Assay Protocol TD-S Date: 2/20/2020
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Materials
 X-Gluc (GoldBio Catalog # G1281)
 Tissue Fixative Buffer
 Extraction Buffer
 Assay Buffer
 STOP Buffer
 MES (GoldBio Catalog # M-091)
 Mannitol
 Dithiothreitol, DTT (GoldBio Catalog # DTT)
 MUG (GoldBio Catalog # MUG)
 dH2O
 Formaldehyde
 Dimethylformamide (DMF)
 Phosphate buffer
 Na2EDTA
 Sodium Lauryl Sarcosine
 Triton X100
 Na2CO3

Preparation of Solutions and Buffers

For MES Stock Solution
 Dissolve 10.66 g MES (GoldBio Catalog # M-091) in 80 ml dH2O.
 Adjust pH with NaOH and fill to 100 ml.
 Store at room temperature.

For Tissue Fixative Buffer

 1.6 ml Formaldehyde (0.6% final concentration).
 4 ml 0.5M MES Stock Solution (20mM final concentration, pH 5.6).
 10.93 g Mannitol
 Fill to 100 ml with dH2O

For 200mM Phosphate Buffer, pH 7.0

 Stock A: 200mM NaH2PO4 (24.00 g/L) in dH2O.
 Stock B: 200mM Na2HPO4 (28.39 g/L) in dH2O.
 For pH 7.0, combine 38 ml Stock A with 62 ml Stock B.

For X-GLUC Stain

 Dissolve 5 mg X-Gluc in 1 ml DMF.
 Add 9 ml of 50mM Phosphate Buffer, pH 7.0.

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GUS Gene Assay Protocol TD-S Date: 2/20/2020
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For Extraction Buffer
 25 ml phosphate buffer 200mM (Final concentration 50mM, pH 7.0).
 0.2 ml 0.5M Na2EDTA (final concentration 1mM).
 1 ml DTT (final concentration 10mM)
 0.33 ml 30 % Sodium Lauryl Sarcosine (final concentration 0.1%)
 1 ml of 10% Triton X-100 (final concentration 0.1%)
 Total volume is 100 ml

Assay Buffer
 Dissolve 17.6 mg MUG in 50 ml extraction buffer in a 50 ml disposable polypropylene
tube (final concentration is 1mM MUG).
 Store at 4°C for up to two weeks.

Stop Buffer
 Dissolve 21.2 g of Na2CO3 in dH2O (final concentration 0.2M).
 Fill to 1 L with dH2O

Method
Histochemical Assay
Currently, the substrate X-Gluc is used for the histochemical localization of β-glucuronidase
activity in tissues and cells. This substrate is highly effective and yields a blue precipitate at the
site of enzyme activity. Notably, there are numerous variables that affect the quality of
βglucuronidase histochemical localization, including all aspects of tissue preparation and
fixation, as well as the reaction itself. Thus, it is necessary to understand the nature of the
reaction. The initial product of glucuronidase hydrolysis of X-Gluc has no color. Instead, the
initial indoxyl derivative produced must undergo an oxidative dimerization to form the
insoluble and highly colored indigo dye. This dimerization is stimulated by atmospheric oxygen,
and can be enhanced by using an oxidation catalyst such as a K+ ferricyanide/ferrocyanide
mixture. Without a catalyst, the results are often very good, but one must consider the
possibility that localized peroxidases may enhance the apparent localization of glucuronidase.
In addition, fixation conditions will vary with the type of tissue and its permeability to the
fixative. For example, if glutaraldehyde is used, one must note that it does not easily penetrate
the leaf cuticle, but it does penetrate stem cross sections well. Alternatively, formaldehyde
seems to be a more gentle fixative than glutaraldehyde and can be used for longer times.
Furthermore, whole tissues, callus, suspension culture cells and protoplasts, whole plants or
plant organs can be stained, but survival of the stained cells is not certain. After staining,
clearing the tissue with 70% ethanol seems to improve contrast in many cases.

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Gold Biotechnology / FM-000008 TD-P Revision 1.0
GUS Gene Assay Protocol TD-S Date: 2/20/2020
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Procedure
1. Take one or two fresh leaf disks directly from a selection plate. Cut fresh disks into
quarters.

2. Transfer sections to 0.5 ml of X-Gluc stain in 24-well plates and incubate for 1 hour to
overnight at 37°C.

3. After staining, rinse sections in 70% ethanol for at least 5 minutes.

Note: If green color persists, clear the tissue of chlorophyll by soaking in 70% ethanol for at
least 4 hours.

4. Examine for GUS stain under dissecting microscope.

Note: Immerse tissue in Tissue Fixative Buffer if fresh tissues are not used.

5. Store in fume hood at room temperature for up to 3 months (or until precipitates
appear).

Fluorimetric Assay

Although various spectrophotometric substrates for GUS are available, GUS activity in solution
is usually measured with the fluorometric substrate 4-methylumbelliferyl-β-D-glucuronide
(MUG). Fluorometry is preferred over spectrophotometry because of its greatly increased
sensitivity and wide dynamic range. The assay is highly reliable and simple to use. Occasionally,
endogenous compounds will interfere with the assay, either by quenching or by producing a
high background fluorescence. In these situations, fluorometric substrates with differing
excitation and emission wavelengths are recommended (the most popular being resorufin-β-
Dglucuronic acid). The substrate 4-trifluoromethylumbelliferyl-β-D-glucuronic acid (4-TFMUG)
allows continuous monitoring of GUS activity because, unlike MUG, it becomes fluorescent
upon hydrolysis at the assay pH. In contrast, after hydrolysis of MUG by GUS, the reaction must
be terminated with a basic solution. This stops the enzyme reaction and causes the
fluorescence.

Procedure
1. Homogenize approximately 100 mg of callus or one fresh leaf disk in 100 µl Extraction
Buffer in a 1.5 ml centrifuge tube. Use a small amount of sand or glass beads in the
mixture.

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2. Centrifuge for 5 minutes at 4°C at 25,000 g. Proceed to Step 1 of the Fluorogenic Assay
Protocol.

Note: Extracts can be stored at -70°C with no loss of activity for a long time, or at 4°C with little
loss of activity. Avoid storage at -20°C, which kills the enzyme in lysis buffer.

Note: If the extract is high in endogenous fluorescent compounds or produces high levels of
polyphenolics, they may be extracted in extraction buffer with polyclar (insoluble polyvinyl
pyrollidone) followed by a brief spin column of Sephadex G 25 to eliminate almost all
polyphenolics and low molecular weight fluorescent contaminants from the extract.

Fluorogenic Assay
Protocol Procedure
1. Incubate 0.5 ml aliquots of Assay Buffer at 37°C to pre-warm the buffer.

2. Add 50 µl of extract to 0.5 ml Assay buffer.

3. Mix thoroughly with pipette tip or vortex.

4. At regular time intervals (30 minutes for high GUS activity or 1 hour to overnight for low
GUS activity) remove successive 100 µl aliquots into labeled 1.5 ml centrifuge tubes
containing 0.9 ml Stop Buffer.

Note: Take 3-4 time points if possible and one overnight incubation.

Typical preliminary results can be obtained by placing the 1.5 ml centrifuge tubes on a UV
transilluminator used for observing stained ethidium stained gels.

Associated Products
 X-Gluc (GoldBio Catalog # G1281)
 MES (GoldBio Catalog # M-091)
 Dithiothreitol DTT (GoldBio Catalog # DTT)
 MUG (GoldBio Catalog # MUG)

Gold Biotechnology
St. Louis, MO
Ph: (314)890-8778
Web: www.goldbio.com 5
Email: contactgoldbio86@goldbio.com
Gold Biotechnology / FM-000008 TD-P Revision 1.0
GUS Gene Assay Protocol TD-S Date: 2/20/2020
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References

Gallagher, S. R. (1992). GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression.
Boston: Academic Press.

Horsch R. B., Fry J. E., Hoffmann N. L. Eichholtz D. Rogers S. G., and Fraley R. T. (1985). A simple
and general method for transferring genes into plants. Science 227:1229 1231.

Janssen, B-J. and Gardner, R. C. (1989). Localized transient expression of GUS in leaf discs
following cocultivation with Agrobacterium. Plant Molecular Biology. 14:61-72.

Jefferson, R. A. (1987). Assaying chimeric genes in Plants: The GUS gene fusion system. Plant
Molecular Biology Reporter, 5:387-405.

Jefferson R. A., Kavanagh T. A., and Bevan M. W. (1987). GUS fusions betaglucuronidase as a
sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 6:3901 3907.

Kim, K., Franceschi, V. R., Davin, L. B., and Lewis, N. G. (2006). β-Glucuronidase as Reporter
Gene: Advantages and Limitations. Arabidopsis Protocols, 263-274. Doi:10.1385/1-
59745003-0:263.

Klee H. J., Horsch R. B., and Rogers S. G. (1987) Agrobacterium mediated plant transformation
and its further applications to plant biology. Annual Review of Plant Physiology, 38:467-486.

Nester E. W., Gordon M. P., Amasino R. M., and Yanofsky M. F. (1984). Crown Gall: A molecular
and physiological analysis. Annual Review of Plant Physiology, 35:387 413.

Gold Biotechnology
St. Louis, MO
Ph: (314)890-8778
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Email: contactgoldbio86@goldbio.com