Hi everyone!
I'm currently optimizing my [35S]GTPγS assay in whole rat brain homogenates and I'm only getting 50% stimulation over basal with 10uM DAMGO. Another thing is that the difference in efficacy between DAMGO and morphine is rather small (148 vs. 125%). Thus, DAMGO doesn't seem to act as a full agonist in my system. When I was running the assay (in a 1 ml format) in another lab Emax values were 190% for DAMGO and 140% for morphine and I could clearly see the difference between the efficacy of both drugs.
My binding buffer composition is as follows: 50mM Tris-HCl (pH 7.4), 3 mM MgCl2, 100 mM NaCl, 1mM EGTA and 30uM GDP. The assay is run in a 250 ul format with 0.05 nM [35S]GTPγS and 15ug/ml of added protein for 1h on a shaker set to 30oC. The samples are filtered with the FilterMate Harvester onto GF/B Unifilter plates and dried for 2 h at 50oC. The scintillation fluid I'm using is Microscint-20 (45ul on each filter). I've already tried different protein, GDP and Mg2+ concentrations with no improvement in efficacy. I'll be running another experiment with different Na+ concentrations next week, but I doubt that'll bring a breakthrough though. Did anyone of you encounter such a problem with your assay? If you have some tips please share.
Anna
Hi Anna!
This is not an error. Indeed, using whole brain homogenate, the maximal stimulation by DAMGO remains about 50% over the basal level. Please note, that it means 150 % "cumulative" stimulation. You are right, that a value around 90% would be better, but it is not easy to reach this level using whole brain homogenate, rather using striatum or cortex-originated membranes. DAMGO is full agonist in your system, believe me, as morphine produced less stimulation, as usual. Pharmacologists do not like this, the prototype drug morphine being as partial agonist, but this is the case...
Hi Sandor!
Thank you so much for your reply. Hoping that you are well. As you probably have guessed already the lab I mentioned was your lab that I visited some time ago to learn the GTPgammaS assay :) In my last experiment, morphine produced slightly less stimulation, but in the one before, the Emax values were identical for both drugs. I was surprised because you mentioned morphine being a partial agonist and DAMGO giving much more stimulation than 50%. Another thing is that I also get 10-times lower EC50's than I got in your lab. I know that the EC50 values are not accurate when determined in GTPgammaS assays due to the presence of sodium, but this inconsistency still worries me a bit. I don't remember which membranes I used when I visited your lab, but I think I'll try the brain regions you mentioned and see how it goes. Do you also use spinal cord homogenates? Do you see a clear distinction between the efficacy of DAMGO and morphine?
All the best,
Anna
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