GTPgammaS Binding Assay

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cbusi
cbusi's picture
GTPgammaS Binding Assay

Hi all,

I am really frustrated. All the GTPgammaS assay I've done have a non-specific binding very high, sometimes more than basal binding. Treatment of agonist have no or little increase in the GTPgammaS binding. have you some advice for me?
Thanks very much
 

R Bishop
R Bishop's picture
cbusi,

cbusi,
Can you give me some details on how you are performing this experiment? Type of cells, how much The [35S]GTPγS (specific activity), buffers, times, etc.  Troubleshooting these types of experiments is all in the details.
My suspicion is you have a low level of comtaimination (bacteria, myco, etc). This would explain the "high" background.

Alternatively, your agonist is not any good and not displacing the GDP from your GPCR of interest. Therefore what you think is high or inconsistent is acutally background binding in both the treated and untreated.
 
Again feed us some details and we'll try and help
 
Rus
 

cbusi
cbusi's picture
Thanks you Rus,

Thanks you Rus,
I use Hek293 cell stably tranfected with D1 receptor, 0.5nM [35S]GTPγS, assay Buffer contains 20mM HEPES pH7.4, 20mM MgCl2, 100mM Nacl, GDP 100uM. 40-60ug of membrane preparation were incubated with or without agonist for 30 minutes at 30°C. Non specific binding was defined using 100uM Gpp(NH)p. I don't know if this compost is good to displace [35S]GTPγS, even if there are some articles that use it, what do you think?   
The agonist is SKF81297 specific for D1 receptor at the concentration of 10uM.
Thanks for your interest
cbusi
 

R Bishop
R Bishop's picture
 Cbusi,

 Cbusi,
No problem, Im happy to help. Its why we created Scientist Solutions!
It is my understanding that [35S]GTPγS cannot be displaced from GPCRs in the presence of Mg.
(Bokoch, 1984. Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Journal of Biological Chemistry 259, 3560 – 3567.)
Have you seen papers that contradict this for dopamine receptors like D1?  
So then the question becomes how to measure non-specific binding to the membranes. Do you run a "non-transfected" HEK293 control? To me that would be the perfect control, then you subtract the non-transfected counts from the transfected counts pre and post agonist treatment and you got your answer as to whether D1 is active etc. 
 
Let me know what you think.
Rus
 

aif
aif's picture
Hi!

Hi!

I'm having a similar problem (sometimes I get high counts, and no response to the agonist), so I was wondering if and how did you solved your problem.

Thanks in advance.

Ana