GDP dissociation assay


1. Preparation of the proteins:


microdialysis: 2 x 1h against exchange buffer (50 mM Tris/HCl pH 7,5, 50 mM NaCl, 1 mM DTT, 5 mM EDTA), 4°C

2. Loading of the GTPase with 3H.GDP

  • mix 17.5 µg GST-GTPase with 3.5 µCi [3H]GDP in 45 µl exchange buffer (with BSA)
  • incubate for 20 min at RT
  • add 45 µl cold SEB to stop the loading, keep on ice
  • dilute loaded GTPase with 750 µl cold RB (total volume: 840 µl, sufficient for 10 assays), you may store the diluted solution for less than 2 h on ice. One assay are two time points with two measurements, = four filters)

3. Exchange assay:

  • The incubation time is 20 min at 25°C. For each time point (t=0 min, t=20 min) the reaction solution is filtered through a nitrocellulose filter to separtate protein wtih bound radioactivity and free radioactivity.
  • prepare filter unit with NC filter soaked in Stop buffer, apply vacuum
  • mix 80 µl [3H]GDP loaded GTPase (final about 0.2-0.3 µM) with the exchange factor (about 0.1 µM). Adjust the reaction volume with RB (without GTP) to 160 µl. Final GTP concentration is 0.5 µM
  • filter immediately 2x35 µl and wash filters with 2x5 ml cold stop buffer

4. measurement

  • incubate the reaction solutions at 25°C for 20 min, filter again 2x35 µl. wach filters with 2x5 ml stop buffer
  • dry the filters, transfer to a scintillation vial, add 2 ml of scintillation liquid and count radioactivity (t=1 min)

Buffer:

Exchange buffer:
50 mM Tris/HCl pH 7.5
50 mM NaCl
5 mM EDTA
1 mM DTT
1 mg/ml BSA
add BSA prior to usage

Stop exchange buffer (SEB)
50 mM Tris/HCl pH 7.5
10 mM MgCl2
1 mM DTT

Reaction buffer (RB)
50 mM Tris/HCl pH 7.5
1 mM GTP
2 mM MgCl2
add GTP prior to usage

GTP
100 mM GTP
store at -20°C

Stop buffer (SB)
50 mM Tris/HCl pH 7.5
10 mM MgCl2


Materials:

Nitrocellulose filters
Schleicher and Schuell, NC45 membrane filters 0.45 µm, 25 mm, Ref. Nr. 10401106

Filtrationstopf, Millipore, mit Gummistopfen und Vakuumanschluß

Microdialysis:
Slide-A-Lyzer, 10 kDa, 10-100 µl, Pierce, Nr. 69572, Vertrieb: Perbio Science, www.perbio.com, Tel.: 0228/9125650, ca. 1€/Filter

[3H]GDP
Amersham TRK335, [8-3H]GDP (426 GBq/mmol, 9,25 MBq, 250 µCi)