GUS Fluorometric Assay

Fluorometric Assay of GUS Activity in Arabidopsis Plants


(Gallagher, 1992; Jefferson et al., 1987)



  • After incubation of plants with inducer of the gene circuit (CK, TNT, etc.), grind plants/leaves in GUS Extraction Buffer (100 mL for excised leaves, 200 mL for whole plants) in a 1.5 mL microfuge tube, using a drill fitted with autoclaved plastic pestles. Keep tubes on ice.


  • Spin down the tubes @ 10,000 rpm in a refrigerated centrifuge for 10-15 min.


  • Transfer the cleared supernatant to a new tube and place it on ice.



GUS Reaction:


MUG + GUS  ®  4-MU


MUG = 4-methylumbelliferyl b-D-glucuronide

GUS = b-glucuronidase (enzyme)

4-MU = 4-methylumbelliferone


  • In a 0.5 mL microfuge tube combine 50 mL of the protein extract obtained above with 50 mL of GUS Assay Buffer (2 mM MUG in GUS Extraction Buffer) .


  • Mix well by pipetting up and down, and centrifuge for 15 sec.


  • Incubate reaction @ 37oC for at least 1 hour (make note of time of incubation for later calculations).


  • Stop reaction by pipetting 50 mL of the reaction mixture into 1.95 mL Carbonate Stop Buffer (0.2 M Na2CO3)  contained in a 4 mL tube (avoid excessive light exposure of tubes because 4-MU is light sensitive).


  • Read 4-MU fluorescence units in a DyNA Quant 200 Fluorometer (Hoefer, Inc., San Francisco, CA) using a 4-MU standard solution to calibrate the instrument. First, zero the instrument with 1.9 mL of Carbonate Stop Buffer, then add 100 mL of the 4-MU standard solution to the stop buffer (see Solutions below), and calibrate it to 500 units.



Total Protein Quantification:


Total protein quantification is done using the Bradford method (Bradford, 1976):

  • Prepare a 5-fold dilution of the concentrated Bradford reagent (Bio-Rad Laboratories, Palo Alto, CA):

                                                            1 part of reagent + 4 parts of diH20


  • Aliquot 20 mL of the protein extract prepared above and BSA standards (see below) into a 1.5 mL microfuge tube.


  • Add 1 mL of the diluted Bradford reagent.


  • Vortex briefly to mix.


  • Incubate reaction @ RT for 10-15 min.


  • Read Absorbance @ 595 nm on a spectrophotometer.


Use BSA (stock 10 mg/mL) to prepare BSA standards:


BSA Conc. (mg/mL)              BSA          +          Extr. Buffer

            0.0                               0 mL          +          100 mL                   = 100 mL

            0.1                               1 mL          +            99 mL                   = 100 mL

            0.2                               2 mL          +            98 mL                   = 100 mL

            0.4                               4 mL          +            96 mL                   = 100 mL

            0.6                               6 mL          +            94 mL                   = 100 mL


Plot the BSA standard curve in Microsoft Excel. For example:




  1. GUS Extraction Buffer

Final conc.        Stock            Vol. for 10 mL

NaHPO4, pH 7.0                     50 mM                          1M                     0.5 mL                              

b-mercaptoethanol                 10 mM                          14.4 M               7 mL

Na2EDTA, pH 8.0                   10 mM                          0.5 M                 0.2 mL

Sarcosyl                                  0.1%                  30%                    33 mL

Triton X-100                           0.1%                  10%                    100 mL

H2O                                         —-                    —-                     9.16 mL


  1. GUS Assay Buffer (2 mM MUG in GUS Extraction Buffer)


Mix 22 mg of 4-methylumbelliferyl b-D-glucuronide (MUG) with 25 mL of GUS Extraction Buffer. Tip: weigh out a small amount of MUG and adjust the volume of GUS Extraction Buffer accordingly.


  1. Carbonate Stop Buffer (0.2 M Na2CO3)


Weigh out 6.2 g Na2CO3.H2O and dissolve it in 250 mL of diH20.


  1. 4-MU standard solution


First, prepare a concentrated MU calibration stock solution (1 mM 7-hydroxy-4-methylcoumarin, MU, in diH2O):

Mix 19.8 mg (0.0198 g) of MU into 100 mL of diH2O to produce a 1 mM solution.

Dilute 10 mL of the 1 mM MU solution into 10 mL of diH2O to make the
1 mM MU stock (store this solution at 4oC, protected from light).


Second, for calibration of the fluorometer, make a 50 nM MU solution, by diluting 100 mL of the 1 mM concentrated MU calibration stock solution prepared above into 1.9 mL Carbonate Stop Buffer. This solution must be made fresh just before use.



Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-254.


Gallagher, S.R. (1992) Quantitation of GUS activity by fluorometry. In GUS Protocols: using the GUS gene as a reporter of gene expression (Gallagher, S.R., ed): Academic Press, Inc., New York, pp. 47-59.


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