GTP Binding Assays in Arabidopsis thaliana    

Materials and Reagents

1. GTP γ³⁵S 1,200 Ci/mmol (PerkinElmer, catalogue number: NEG030H250UC )
   
2. 0.45 μm nitrocellulose filters (Bio-Rad, catalogue number: 162-0115 )
   
3. Scintillation solution (Optiphase Hisafe 3, Perkin Elmer Inc, catalogue number: 1200-437 )
   
4. Bradford reagent
5. Triton X-100
   
6. Tris HCl (pH 8)
   
7. NaCl
   
8. EDTA
   
9. DTT (USB Corporation, Cleaveland OH USA, cataslogue number: 15397 )
   
10. Roche complete protease inhibitor (Roche, Molecular Biochemicals)
    
11. Extraction buffer (see Recipes)
    
12. Reaction buffer (see Recipes)
13. Initiation buffer (see Recipes)
    
14. Stop buffer (see Recipes)

Equipment

1. Liquid Scintillation Counter Wallac 1214 RackBeta (Pharmacia, Turku, Finland)

Procedure

I.  Protein extraction protocol (Keep all steps at 4 °C)

1. Harvest approximately 300 mg of Arabidopsis tissue in liquid nitrogen.
   
2. Homogenize with mortar and pestle to a fine powder.
   
3. Resuspend the powder in 500 μl of extraction buffer (you can use manual homogeneizer).
   
4. Centrifuge 15 min at 10,000 rpm at 4 °C and discard the pellet.
   
5. Measure protein concentration in the supernatant as described (Bradford, 1976).
   

II.  GTP binding assay (reaction: final volume 200 μl)

1. Mix 100 μg of proteins in 100 μl extraction buffer with 80 μl of reaction buffer.
   
2. Add 20 μl of Initiation Buffer.
   
3. Expose to the corresponding stimuli (Light, agonist etc).
   (You should adjust the specific stimuli, time of exposition, concentration, intensity etc where you can detect optimum binding).
   
4. Incubate the reaction 10 min at room temperature.
   
5. Add 2 ml of ice-cold stop buffer to finish the reaction.
   
6. Filter the reaction through 0.45 μm nitrocellulose filters using vacuum. We use 3 cm diameter disks.
   
7. Wash the disk filters 5 times with 2 ml of cold Stop Buffer using vacuum. This step is important to wash away unbound GTP γ³⁵S.
   
8. Dry disk filters containing the membranes for 15 min at 75 °C.
   
9. Put the disks in plastic vials containing 2 ml of scintillation solution.
   
10. Record the cpm in the ³⁵S energy range with a liquid scintillation counter.
    
11. Important: Unspecific binding must be estimated using blank reactions. We recommend three replicates of two possible blanks: reactions without proteins (reaction buffer plus initiation buffer) or 100 μg of protein samples preheated to 95 °C for 10 min. We did not find differences in GTP binding between both blanks.
    
12. Subtract the average cpm obtained with the blank samples from the cpm obtained for each measured sample to obtain the correct value.
    

Recipes

1. Extraction Buffer
   50 mM Tris HCl pH 8
   100 mM NaCl
   1 mM EDTA
   1 mM DTT
   0.1% Triton X-100
   1x Roche complete protease inhibitor
   
2. Reaction buffer (use 80 μl from the stock per reaction)
   

   Stock	Final concentration in 200 μl reaction
   50 mM Tris HCl (pH 8)	20 mM Tris HCl (pH 8)
   250 mM NaCl	100 mM NaCl
   2.5 mM EDTA	1 mM EDTA
   2.5 mM DTT	1 mM DTT
   0.25% Triton X-100	0.1% Triton X-100

3. Initiation buffer (use 20 μl from the Stock per reaction) (pH 8)
   

   Stock	Final concentration in 200 μl reaction
   100 mM MgCl2	10 mM MgCl2
   1% Triton X-100	0.1% Triton X-100

   Add GTP γ³⁵S, 300,000 cpm/reaction.
   
4. Stop buffer (use 2 ml per reaction)
   20 mM Tris-HCl (pH 8)
   100 mM NaCl
   25 mM MgCl₂
   1 mM phosphate buffer
   

Acknowledgments

This protocol was adapted from Fox et al. (2012). This work was financially supported by FonCyT PICT 2010 num 1821 to A.M.

References

1. Fox, A. R., Soto, G. C., Jones, A. M., Casal, J. J., Muschietti, J. P. and Mazzella, M. A. (2012). cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis. Plant Mol Biol 80(3): 315-324.