Quantitative Gus Activity Assays

This protocol was contributed by Miguel Blazquez (Salk Institute).

GUS activity can be accurately determined both in extracts and in intact plant Lssue us ng 4-methylumbellyferyl p-D-glucuronide (4-MUG) as a substrate (Jefferson et al. 1987). Upon hydrolysis by GUS, the fluo-rochrome 4-methyl umbelliferone (4-MU) is produced. Using excitation at 365 nm, and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected (see below). GUS activity can be measured either in plant extracts or in whole-plant tissue samples.

GUS Activity Assay of Plant Extracts Materials

Spectrofluorometer (e.g, LS 45 Luminescence spectrometer [Perkin

Elmer] or SPECTRAmax Gemini XS [Molecular Devices])

Liquid nitrogen

GUS extraction buffer

50 mm sodium phosphate (pH 7.0) 10 mm EDTA (pH 8.0) 0.1% SDS 0.1% Triton X-100

Keep at room temperature. Just before use, add 10 mm (3-mercap-toethanol and 25 |ig/ml PMSE

4-MUG (25 mM in GUS extraction buffer) (Rose Scientific)

Sodium carbonate (1 M)

Plastic micropestles (VWK)

CAUTION: p-mercaptoethanol, GUS, liquid nitrogen, PMSF, SDS, sodium carbonate (see Appendix 3)


1. Collect 200-500 mg of tissue in a 1. 5-ml microcentrifuge tube. Freeze immediately in liquid nitrogen. Store at -80°C if necessary.

2. Use a chilled plastic micropestle to grind the tissue to a fine powder; making sure that the tissue always remains frozen.

3. Add 150 pi of GUS extraction buffer and store in liquid nitrogen while processing the remaining samples.

4. Centrifuge at 15,000 rpm for 10 minutes in the cold in a microcentrifuge.

5. Transfer the supernatant (crude extract) to a fresh microcentrifuge tube and keep on ice.

6. Prepare reaction mix containing 1 mM 4-MUG in GUS extraction buffer. Aliquot 1 ml of the reaction mix, for each sample, into a microcentrifuge tube and prewarm to 37°C.

7. For each sample, prepare two glass vials containing 900 pi of 1 M sodium carbonate, the stop reagent.

8. Add 10 pi of crude extract (see Step 5) to the reaction tubes, at 30-second intervals, up to 15 samples per run.

Fast workers can process samples at shorter intervals, but 30 seconds is normally appropriate. With more than 15 samples, a second set should be started after the first set of 15 samples is finished.

9. After exactly 10 minutes, transfer a 100-pl aliquot of each reaction to the first corresponding vial with stop reagent. Continue at 30-second intervals.

10. After 20 minutes, transfer another 100-pl aliquot of each reaction to the second vial with stop reagent.

11. Prepare the 4-MU standard curve by diluting the 4-MU stock to 100 nM, 250 nM, and 500 nM in stop reagent (1 M sodium carbonate).

The exact concentration range might be different depending on the sensitivity of the fluorometer used.

12. Measure the fluorescence of these solutions under the following conditions and plot a standard curve of fluorescence against concentration.

Excitation wavelength 365 nm Emission wavelength 455 nm Filter wavelength 430 nm

If the fluorometer does not allow the use of these particular wavelengths, select the closest available values or perform a wavelength scan on a 4-MU standard solution. The filter prevents the recording of em si 'ons below 430 nm, which might correspond to nonhy-drolyzed 4-MUG.

13. Measure the fluorescence of each set of samples and use the standard curve to calculate the amount of 4-MU per ur it of t?me (i.e., nmoles min"1 ml"1) for each sample.

14. Determine the total protein concentration in each sample, using any conventional method (e.g., that of Bradford 1976).

15. Calculate GUS activity in (nmoles 4-MU) min"1 (mg protein)-1.

GUS Activity Assay in Intact Tissue

This method is based on the permeability of both 4-MUG and 4-MU through plant tissue, and it consists of the incubation of the tissue with the reagent and the quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be normalized per unit tissue weight, per unit protein (if it is determined in the tissue after the incubation with 4-MUG), or per sample. The latter is particularly useful if GUS is expressed only in a subset of cells within the tissue assayed. The advantage of this method is that many samples can be assayed with relatively little effort, so it is particularly appropriate for large-scale screens. For this reason, the following protocol has been adapted for the use of 96-well microtiter plates (200-|xl wells). It requires a fluorometer with a microplate reader.


GUS extraction buffer (as above, but PMSF or P-mercaptoethanol is not necessary) 4-MUG (25 mM, Rose Scientific) in GUS extraction buffer 4-MU (10 mM, Sigma) in water, as standard Sodium carbonate (1 M), as stop reagent Fluorometer with microplate reader

CAUTION: p-mercaptoethanol, PMSF, sodium carbonate (see Appendix 3)


1. Prepare the reaction mix containing 1 mM 4-MUG in GUS extraction buffer. Pipette 100 pi into each microtiter plate well.

2. Harvest tissue (e.g., whole seedling <10 days old, one cotyledon, the apex, flower cluster, etc.) and submerge in solution.

3. Incubate the plate at 37°C.

The incubation time will depend on the strength of the promoter that drives GUS expression. For weak promoters, an incubation time of up to 16 hours may be required. It is important to determine the optimal incubation time the first time that a particular line is used, since it must be long enough to provide measurable amounts of fluorescence, but remain within the linear range of the reaction (i.e., not all 4-MUG has been consumed).

4. Stop the reaction by adding 50 pi of 1 M sodium carbonate to each well.

5. Prepare the 4-MU standard curve by diluting the 4-MU stock to 100 nM, 250 nM, and 500 nM in stop reagent.

The exact concentration range might be different depending on the sensitivity of the fluorometer used.

6. Transfer a 100-pl aliquot from each well to a new microtiter plate. Measure fluorescence as described above, and calculate GUS activity in each sample.

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