Hi all,

I am really frustrated. All the GTPgammaS assay I've done given reverse results. That is, treatment of agonist decreases the GTPgammaS binding. Do you have any idea?
If so, can I share you GTPgammaS binding assay protocol?
Attached is my detailed method.

Thanksssssssssssssssss!

Maybe you can look at these reviews:
1) The [35S]GTPgS binding assay: approaches and applications
in pharmacology C. Harrison, J.R. Traynor - Life Sciences 74 (2003) 489508

2) Principles: Extending the utility of
[35S]GTPgS binding assays
Graeme Milligan TRENDS in Pharmacological Sciences Vol.24 No.2 February 2003

However, you can also consider that you are dealing with an inverse agonist. Sometimes inverse agonism at GPCRs may be easily observed in the absence of Na+ ions. I don't know whch kind of protocol you are using.

Furthermore, there is also Lazareno S. Methods in Molecular Biology Vol 106 1999 231245

Thanks a lot. I did red these reviews. But still can not figure out it.
Anyway, thanks a lot. ,,,,,,,,???

Hi Xun, here is our membrane preparation protocol for GTP-gamma binding assay:

Membrane preparation for GTP-g binding assay (293 and cos cells)

1. Wash10 cm plates with cells with 5 ml of cold PBS w/o Ca+2 or Mg+2 twice.
2. Add 5 ml of cold lysis buffer (10 mM Tris-HCl pH7.4, 1 mM EDTA with B&M protease inhibitor cocktail tablet).
3. Set on ice for 10 min.
4. Scrape the cells into a Dounce homogenizer, and break the cells with 25 strocks.
5. Spin at 1000 RPM for 5 at 4oC
6. Transfer the Sup to sorvall-34 tubes and spine at 16,000 RPM for 30 min.
7. Discard the sup, and re-suspend the pellets in 1 ml of 1 x binding buffer (75 mM Tris-HCl pH 7.4, 12.5 mM MgCl2, 1mM EDTA with proteinase inhibitors) by passing through a syringe attached to a 25 G needle a few time.
8. Add another 0.5 ml binding buffer with protease inhibitors, mix. aliquot and store at 80 oC.

I hope it helps.