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Signaling through G proteins constitutes an ancient mechanism that functions in the transduction of extracellular signals into intracellular responses. Activation of a proper receptor by a stimuli leads to an exchange of GDP for GTP, the activation of G proteins and the dissociation of Gα-GTP from the Gβγ dimer for the heterotrimeric G proteins. The G protein subunits remain active until the intrinsic GTPase activity or/and a GTPase activating protein result in the hydrolysis of GTP to GDP and the inactivation of the protein. Here we describe a protocol for measuring GTP binding activity from Arabidopsis plant protein extracts using GTP γ35S. GTP γ35S assay measures the level of G protein activation following a stimuli, by determining the binding activity of the non-hydrolysable analog GTP γ35S. To determine specific G protein activities specific mutants and/or overexpressors extracts should be included and measured as controls.

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GTP Binding Assays in Arabidopsis thaliana

Biochemistry > Protein > Activity
Authors: Ana Romina Fox
Ana Romina FoxAffiliation: Plant Molecular Biology, INBEBI, Buenos Aires, Argentina
Bio-protocol author page: a594
 and Maria Agustina Mazzella
Maria Agustina MazzellaAffiliation: Plant Molecular Biology, INBEBI, Buenos Aires, Argentina
For correspondence: mazzella@dna.uba.ar
Bio-protocol author page: a595
Vol 3, Iss 9, 5/5/2013, 2745 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.752

[Abstract] Signaling through G proteins constitutes an ancient mechanism that functions in the transduction of extracellular signals into intracellular responses. Activation of a proper receptor by a stimuli leads to an exchange of GDP for GTP, the activation of G proteins and the dissociation of Gα-GTP from the Gβγ dimer for the heterotrimeric G proteins. The G protein subunits remain active until the intrinsic GTPase activity or/and a GTPase activating protein result in the hydrolysis of GTP to GDP and the inactivation of the protein. Here we describe a protocol for measuring GTP binding activity from Arabidopsis plant protein extracts using GTP γ35S. GTP γ35S assay measures the level of G protein activation following a stimuli, by determining the binding activity of the non-hydrolysable analog GTP γ35S. To determine specific G protein activities specific mutants and/or overexpressors extracts should be included and measured as controls.

Keywords: GTP binding, Arabidopsis, GPA1

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.


How to cite this protocol: Fox, A. R. and Mazzella, M. A. (2013). GTP Binding Assays in Arabidopsis thaliana. Bio-protocol 3(9): e752. DOI: 10.21769/BioProtoc.752; Full Text



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