Views
Assay Optimization
From Assay Guidance Wiki
Contents |
Membrane protein/well and (GDP)
Using a starting buffer such as listed under assay buffer above, determine the optimal amount of membrane protein per well from 5 to 50 μg in the presence of varying concentrations of GDP (guanosine diphosphate) from 0 – 10 μM for transfected cell membranes and from 0 up to 300 ?M for native tissue membranes using a concentration of 200 –500 pM GTPγ35S. Note that Gi/o coupled receptors will require higher concentrations of GDP than Gs or Gq coupled receptors which may give optimal signals in the absence of added GDP. Figure 1 illustrates the marked difference in GDP requirement for determination of muscarinic agonist-stimulated GTPγS binding in rat brain striatal membranes measured by anti-Gq/11 (M1 receptor) versus anti-Go (M4 receptor).
Figure 1. Difference in [GDP] required for Gq versus Go coupled GPCR’s
Effect of Mg++
Determine the optimal Mg++ concentration for the best signal to noise over the range of 1 mM to 10 mM. Figure 2 shows the variation of Mg++ on agonist-stimulated GTPγS binding mediated by GPCR receptors in rat striatal membranes.
Figure 2. Agonist-stimulated GTPγS binding in brain membranes
Effect of NaCl
Determine the optimal amount of NaCl for best signal to noise over the range of 0 – 200 mM. Although 100 mM NaCl is commonly used in these assays note that at times better agonist stimulation may be achieved at lower Na++ and if higher constituitive activity is desired (for evaluating inverse agonists) lowering Na++ will likely provide the best opportunity. Figure 3 demonstrates the effect of NaCl on the constituitive activity of an orphan GPCR.
Figure 3. Optimization of NaCl to measure constituitive activity of an orphan GPCR
Effect of Saponin
The effect of adding saponin at 3 – 100 μg/ml can be explored, but recognize that while saponin may increase signal to background, it may also compromise the quality of concentration response curves. Figure 4 demonstrates the optimization of saponin to achieve the highest signal to background for an orphan receptor where constituitive activity was measured to allow evaluation of inverse agonists. Figure 5 shows how saponin may compromise the quality of some concentration response curves.
Figure 4. Optimization of saponin to measure constituitive activity of an orphan GPCR
Figure 5. Effect of saponin on agonist concentration response curves for some receptor subtypes
Incubation Time
The optimal incubation time for the best signal to background may be determined, but thirty minutes is usually satisfactory for cell membranes and one hour for native tissue membranes.
Antibody dilution for antibody capture assays</H4> If using the antibody capture method the optimal dilution will have to be determined for each lot of antibody. Figure 6 below illustrates the effect of various dilutions of anti-Gs/olf on GTPγS binding mediated by a specific GPCR receptor.
Figure 6. Effect of antibody dilution on basal and agonist-stimulated binding
The use of experimental design and JMP analysis for assay optimization</H4> Experimental design and JMP analysis are convenient tools for optimizing a variety of conditions in a small number of experiments and determining if there are any interactions among the factors. Figure 7A shows an example in which four factors were optimized in a single experiment. Figure 7B shows the two factor interaction profiles from JMP analysis. Parallel lines indicate no interaction and intersecting lines indicate interactions. For instance in this experiment there is virtually no interaction between NaCl and saponin, but there is a significant interaction between the amount of protein and GDP concentration.
Figure 7A. Experimental Design with 4 factors (GDP, Saponin, NaCl and Membrane protein)
Figure 7B. Interaction Plots for Experimental Design (using JMP)
Signal window and Z’ factor
Determine the signal window for the assay under the optimal conditions by running background and maximal stimulation multiple times across assay plates on separate days. Calculate the Z’ factor for the assay using the formula:
A Z’ factor of > 0.5 indicates a useful assay. GTPγS binding assays can be quite reproducible and will give reliable results when signals are greater than 40-50% over background. Even with smaller signals, one can generate reliable concentration response curves by using 4 to 8 replicates per data point.
Evaluation of standard compound concentration response curves
After determining optimal conditions for the assay concentration response curves should be run for standard agonists and antagonists to determine variability and comparability to literature values if available. Most assays will require duplicate determinations per concentration but with exceptional signals one may be able to use single data points for each.
Choice of whole membrane versus antibody capture
Good assays for Gi/o may be developed using whole membranes and WGA beads. Use of antibody capture for Gi/o coupled receptors, however, may reduce assay variability. For Gq and Gs coupled GPCR’s, the antibody capture assay will most likely be superior since most cells and tissues are dominated by inhibitory G-proteins and it is often not possible to develop reliable signals without the antibody technique unless receptors are fused to Gs or Gq (3, 13).
