GTPgammaS Binding Assay

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xun
xun's picture
GTPgammaS Binding Assay

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!

val
val's picture
Maybe you can look at these

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

xun
xun's picture
Thanks a lot. I did red these

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

kumar
kumar's picture
Hi Xun, here is our membrane

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.

saswati1
saswati1's picture
Thanks Kumar for the

Thanks Kumar for the information.

Saurabh 5555
Saurabh 5555's picture
Hi I have faced the same

Hi I have faced the same problem and I got over it after a long time........I tell you something important.......First of all are you using whole mice brain.........I was using whole mice brain first but then I read some literature and only isolated cerebellum............You can also add Adenosine deaminase to this membrane preperation as it will lower basal binding .......You will get good result try this out

val wrote:

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

ripalamin
ripalamin's picture
hello,

hello,

anyone know about homogenate assay under gtpys binding assay..what that means i really dont know?

Need reply ..thanking you,

Ripal

vandna.prasad
vandna.prasad's picture
hi,

hi,
i am facing a similar problem. the ligand behaves as an agonist in the functional assay (calcium release) whereas in GTP binding assay  i get a decrease in signal in comparison to untreated sample. dont know how to interpret it. can anybody please help me out with the assay. i am using cell line overexpressing my receptor of interest.
thanks.

bhawanagupta
bhawanagupta's picture
Hi, May I know which

Hi, May I know which recombonant cell line you r using

vandna.prasad
vandna.prasad's picture
hi,

hi,
i have used CHO-NFAT Gqi5 alpha cell line as a host to over express my gene of interest. cant reveal the name of the receptor but its a GPCR reported to function both through Gi and Gq pathway. The modified host allows all the signaling through Gi to be directed through Gq pathway and hence calcium response.
hope you have some solution for my problem.
thanks

bhawanagupta
bhawanagupta's picture
HI,

HI,
I have some tips for this assay, it may be useful for u.
1. Buffer selection depends on receptor type.
2. U can add EDTA/ saponin/ both in assay buffer
3. U can use sigma BSA(0.2%), but again it depends on type of agonist and receptor
4.Try out lower conc of GDP, u can start with 5, 10 15, 20 & 40 µM ( in most case 5µM gives best result)
5. 100pM radioligand [3H] GTP gammas gives enough signal. I use 50pM in my assay
6. I have always use membrane - 5µg/well and every time it worked.
Try out these parameter. If again you dont get expected results, then send me urs detail protocol, may be I can help you out.
 

vandna.prasad
vandna.prasad's picture
hi,

hi,
thanks for the recommendations...
we essentially use tris EDTA buffer containing sucrose (as mentioned in the kit). what we use is not radiolabelled GTPgamma but Europium labeled GTP (delfia kit from perkin elmer). We did try different amount of protein and GDP and optimized it to right levels.
my problem has been inverse results in this assay in comparison to the functional calcium assay. i can give you the exact protocol to go through.

rsportsman
rsportsman's picture
This may be useful:http://www
bhawanagupta
bhawanagupta's picture
Hi,

Hi,
I have standardized GTP gamma binding assay with S35 radioligand, but not by using DELFIA kit. Sorry I dont have any exp. on this kit.

Brooke
Brooke's picture
Hi bhawanag,

Hi bhawanag,
Do you know what is the function of EDTA? I have a protocol with EGTA added. However, I could not get good window after optimizion of all the parameters, such as GDP, protein, NaCl, MgCl, Saponin. Thank you very much.

LK
LK's picture
 I am having problems with

 I am having problems with GTPys assays too. I used to get around a 100fmol/mg GTPys binding with my agonist and now I dont have more than 40fmol/mg binding levels. I have checked the receptor expression and its good. I dont know what is really wrong. Does anyone have any idea?