ARNO Protein: GEF (Sec7) domain (aa31- 267, human recomb., 6xHis tagged)

On Arrival: 4°C
Before reconstitution, briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 5 mg/ml with ice cold nanopure water (20 ul water per 100 ug protein).When reconstituted, the protein will be in the following buffer: 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2, 5% (v/v) sucrose and 1% (v/v) dextran. In order to maintain high biological activity of the protein it is strongly recommended that the protein solution be supplemented with DTT to 1 mM final concentration, aliquoted into "experiment sized" amounts, snap frozen in liquid nitrogen and stored at -70C. The protein is stable for six months if stored at -70C. The protein must not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated (< 10% humidity) for one year. Further ARNO dilutions should be made in 20 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl₂(not supplied).

ARNO is a guanine exchange factor for Arf family proteins (1). It’s intracellular function is to regulate Arf proteins by transducing the upstream signal from phospholipids, kinases and Arf proteins themselves (2). Arno functions in large concerted cellular events that require re-organization of membranes e.g. cell migration (3, 4), phagocytosis (5), macropinocytosis (6), and insulin controlled organism level growth (7).
Study inhibitors of ARNO GTP/GDP exchange activity
Identification of ARNO Sec7 domain binding proteins
Study of ARNO GEF activity with different GTPases

The biological activity of GE07 can be determined from its ability to catalyze nucleotide exchange on Arf proteins using the nucleotide exchange assay of GDP for mant-GTP. The exchange reactions can be performed by adding EDTA or Arf GEF h.s. ARNO protein to a reaction hence providing a control condition for exchange (see kit Cat. # BK100). The reaction is monitored by fluorescence measurement at 485nm Ex / 525nm Em (bodipy-FL-GDP). Stringent quality control ensures that the exchange rate of Bodipy-GDP or Mant-GDP is enhanced two fold in the presence of 1 µM ARNO. Exchange rates are similar to published examples of ARNO catalyzed exchange in >1 mM Mg2+ containing buffer (Bourgoin et al. 2002, see Figure 2) and with <1 µM Mg2+ containing buffer (Beraud-Defour et al. 1998).

1. Place ARNO vial on ice and dilute to 0.30 ug/ul (8 uM) with ice cold Exchange Buffer.

2. Dilute Arf protein to 1.25 ug/ul (50 uM) with ice cold Exchange Buffer.

3. Add the following components together in a fresh 15 ml Falcon tube and mix well by pipetting or gentle vortex:

Component ul per well

Exchange Buffer 75
50 uM?Arf 5
8 uM ARNO 10

Note: For a total mixture volume, multiply the volume of reagents per well by the number of wells in the experiment, plus add 20% volume for pipetting losses.

4. Incubate for 20 min at room temperature (RT).

5. Lock in the nucleotide by adding 10 ul (per well) of 50 mM MgCl₂.

6. Set up the fluorimeter with Excitation wavelength at 485 nm +/-15 nm and emission wavelength at 525 nm +/- 15 nm at RT.

7. Aliquot the pre-loaded mixture to the assigned wells and place the plate in the fluorimeter.

8. After 5 cycles (150 seconds), place the program on Hold or Pause, and remove the plate.

9. Pipette 10 ul of a) 5 mM GTP solution, b) a small compound, c) a test protein, d) 4 mM EDTA (+ve exchange control) or e) Dilution Buffer (negative control) in respective wells and immediately pipette up and down twice and resume reading for 20 minutes.

10. Save the readings after the kinetic protocols are finished. The exchange rate can be calculated by reducing the data to max slope (using 12 pts) or Vmax with the software that accompanies the plate reader. The exchange curve can be achieved by export to Microsoft Excel.

CS-GE06 SDS-PAGE Analysis. A 20 ug sample of recombinant GE07 (molecular weight approx. 33 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein quantitation was determined using the Precision Red Protein Assay Reagent (Cat. # ADV02). ?Mark12 molecular weight markers are from Life Technologies Inc