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This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which is an antagonist binding to D2, D3, and D4 receptors with comparable affinity. A saturation assay can be used to determine the affinity of a radioligand to a receptor (Kd) and to determine the total number of receptors present in the assay (Bmax). If saturation binding experiments are performed in the absence and presence of a fixed concentration of another, not radiolabeled ligand, it can also be determined whether the other ligand acts in a competitive manner. If the specific radioactivity is low (tritiated) relative to the affinity of the radioligand (< 1 nM), a high assay volume (≥ 1 ml) is required to avoid ligand depletion; this is of particular importance if a receptor source with high expression density is used (e.g. expressed recombinant receptors). To obtain reliable estimates of these parameters at least 6 different concentrations of radioligand must be tested, but particularly when a receptor is first detected in a given tissue or cell type a greater number of concentrations are helpful. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Each experiment can be divided into different steps such as assay preparation, membrane preparation, incubation, filtration, counting of the samples and data analysis. To minimize experimental error all assays are performed at least in duplicate. Radioligand dilutions should be prepared to cover the desired concentration range. Optimally these concentrations should cover both the high range (corresponding to 5-10x Kd and hence saturation of the receptor) and the low range (around Kd), so that both Kd and Bmax can reliably be estimated without undue extrapolation. At each radioligand concentration total and non-specific binding should be determined; the agent used for the definition of non-specific binding (NSB) should be chemically (different family) and physically (avoid combination of two lipophilic compounds) distinct from the radioligand to avoid artifacts. For discussion of specific benefits of chosen assay conditions see van Wieringen et al. (2013) (copy can be obtained from the author).

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[3H]-Spiperone Saturation Binding to Dopamine D2, D3 and D4 Receptors

Neuroscience > Sensory and motor systems
Authors: Jan-Peter van Wieringen
Jan-Peter van WieringenAffiliation: Nuclear Medicine, Academic Medical Center, Amsterdam, Netherlands
Bio-protocol author page: a927
 and Martin C. Michel
Martin C. MichelAffiliation: Pharmacology, Johannes Gutenberg University, Mainz, Germany
For correspondence: marmiche@uni-mainz.de
Bio-protocol author page: a928
Vol 3, Iss 20, 10/20/2013, 2469 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.945

[Abstract] This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which is an antagonist binding to D2, D3, and D4 receptors with comparable affinity. A saturation assay can be used to determine the affinity of a radioligand to a receptor (Kd) and to determine the total number of receptors present in the assay (Bmax). If saturation binding experiments are performed in the absence and presence of a fixed concentration of another, not radiolabeled ligand, it can also be determined whether the other ligand acts in a competitive manner. If the specific radioactivity is low (tritiated) relative to the affinity of the radioligand (< 1 nM), a high assay volume (≥ 1 ml) is required to avoid ligand depletion; this is of particular importance if a receptor source with high expression density is used (e.g. expressed recombinant receptors). To obtain reliable estimates of these parameters at least 6 different concentrations of radioligand must be tested, but particularly when a receptor is first detected in a given tissue or cell type a greater number of concentrations are helpful. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Each experiment can be divided into different steps such as assay preparation, membrane preparation, incubation, filtration, counting of the samples and data analysis. To minimize experimental error all assays are performed at least in duplicate. Radioligand dilutions should be prepared to cover the desired concentration range. Optimally these concentrations should cover both the high range (corresponding to 5-10x Kd and hence saturation of the receptor) and the low range (around Kd), so that both Kd and Bmax can reliably be estimated without undue extrapolation. At each radioligand concentration total and non-specific binding should be determined; the agent used for the definition of non-specific binding (NSB) should be chemically (different family) and physically (avoid combination of two lipophilic compounds) distinct from the radioligand to avoid artifacts. For discussion of specific benefits of chosen assay conditions see van Wieringen et al. (2013) (copy can be obtained from the author).

Keywords: Dopamine, Receptor, Ra

Materials and Reagents

  1. Radioligand [3H]-spiperone (e.g. PerkinElmer, catalog number: NET565250UC)
  2. Butaclamol (e.g. Sigma-Aldrich, catalog number: 55528-07-9)
  3. Receptor-containing membrane suspension
  4. Whatman GF/C filters (e.g. PerkinElmer, catalog number: 6005174)
  5. Poly(ethyleneimine) solution (PEI) (e.g. Sigma-Aldrich, catalog number: 9002-98-6)
  6. Scintillation cocktail (e.g. PerkinElmer, catalog number: 6013641)
  7. Tris-HCl
  8. KCl
  9. CaCl2
  10. MgCl2
  11. Destilled water
  12. Assay buffer (see Recipes)
  13. Wash buffer (see Recipes)
  14. NSB solution (see Recipes)
  15. Radioligand solutions (see Recipes)
  16. Radioligand Dilutions (see Recipes)

Equipment

  1. 96 well plates (polysterene)
  2. Cell harvester (e.g. PerkinElmer)
  3. Ultra-Turrax® (IKA, model: 0001602800) or similar disperser
  4. Water bath
  5. Scintillation counter

Software

  1. Prism (Graphpad Software, San Diego, CA, USA) or similar

Procedure

  1. Before the assay
    1. Prepare assay and wash buffer.
    2. With some receptor sources PEI-pretreated filterplates may improve the TB/SB ratio: Prepare 0.1% PEI solution and pipet 100 μl/filter on the filterplate, subsequently place in refrigerator (4 °C) for at least 2 hours.
    3. The experiment is performed in 96 well plates (polysterene) with a total assay volume of 1,000 μl (450 μl assay buffer + 200 μl radioligand + 200 μl of assay buffer or 5 μM (+)-butaclamol + 150 μl membrane preparation).
    4. Prepare NSB solution.
    5. Prepare radioligand solutions.
    6. Membrane preparation.
      Prepare receptor-containing membrane suspension according to preparation protocol. Re-homogenize suspension in small volume (< 2 ml) using short burst of Ultra-Turrax. Dilute to desired protein concentration and to yield a total volume of about 4 ml per 24 data point experiment. The protein concentration of the membrane suspension should be chosen so that a robust specific binding signal is obtained but at the same time total binding should be < 10% (even better < 5%) of free radioligand concentration. Protein content can be assayed by a variety of essays, e.g. Bradford (1976). Prepare the membrane suspension initially in ice.
    7. Pre warm all solutions for 15 min in 25 °C water bath.
    8. Final preparation (Table 1), add components to wells in following order:
      1. 450 μl assay buffer.
      2. 200 μl assay buffer (TB) or 200 μl 5 μM butaclamol (NSB).
      3. 150 μl membrane suspension.
      4. Start reaction by adding 200 μl radioligand.

        Table 1. Pipetting scheme for sample on microtiter plate
        T1
        T1
        T2
        T2
        T3
        T3
        T4
        T4
        T5
        T5
        T6
        T6












        TB1
        TB1
        TB2
        TB2
        TB3
        TB3
        TB4
        TB4
        TB5
        TB5
        TB6
        TB6
        NSB1
        NSB1
        NSB2
        NSB2
        NSB3
        NSB3
        NSB4
        NSB4
        NSB5
        NSB5
        NSB6
        NSB6

  2. During the assay
    1. Incubate for 120 min at 25 °C in water bath.
    2. Terminate reaction by rapid vacuum filtration over Whatman GF/C filters using a cell harvester. Wash filter 10 times with ice-cold wash buffer.

  3. After the experiment
    1. Add aliquots of 50 μl radioligand to wells of counting plate to determine total radioactivity.
    2. Dry, e.g. in oven for 2 h.
    3. Place sticker on bottom plate. Add scintillation cocktail (20 μl) to filters, place sticker on top of plate and count in a scintillation counter, allow adequate time (15 min) before counting samples.

  4. Data analysis
    The following calculations are required to derive Kd and Bmax from the data.
    1. Carefully inspect raw data for consistency of replicates. Do not light-heartedly eliminate apparent ‘outliers’ from the data set.
    2. Subtract mean non-specific binding from each replicate of total binding to obtain specific binding.
    3. Transform totals to molar concentration of radioligand in the assay, and measured bound values (total binding, non-specific binding and specific binding) to molar amount of ligand based upon the specific radioactivity of the radioligand and the efficiency of the scintillation counter. A representative experiment with D2 receptors may look like this.



    4. Plot specific binding (y-axis) vs. concentration of radioligand (x-axis).
    5. Analyze data by non-linear iterative curve fitting using a rectangular hyperbolic function using one of many available iterative curve fitting programs, e.g. Prism.
    6. Molar amount of Bmax can be corrected for tissue content, mostly the amount of protein per well (mostly yielding fmol/mg protein); alternative normalization e.g. for tissue wet weight, number of cells or DNA content are possible.
    7. As internal quality check, determine the fraction of total binding from total radioactivity in the assay. This needs to be < 10% (better < 5%) as otherwise non-equilibrium conditions may exist. In the latter case, membrane concentration in the assay can be reduced if the measured signal remains robust. Alternatively, assay volume can be increased as this will increase total amount of radioligand (concentration constant) but not amount bound (membrane protein amount constant).
    8. In the past when no computer assisted data analysis was available Kd and Bmax were estimated with the Rosenthal plots, better known as Scatchard plots (Rosenthal invented them but Scatchard made them famous). In these plots the specifically bound radioligand is on the x-axis and is plotted against the ratio of bound/unbound radioligand on the y-axis. The x-intercept corresponds to Bmax and the negative reciprocal of the slope corresponds to Kd. Rosenthal/Scatchard plots can still be useful to visualize that data points indeed fall on a linear line but the estimates derived from this are less reliable than those from iterative curve fitting (the assumptions of linear regression used in these plots don’t meet because x and y are not independent of each other), making this an outdated analysis.
    9. If saturation binding experiments are performed in the absence and presence of an inhibitor, the data can be used to test for a competitive nature of the inhibitor. This is the case if the presence of inhibitor changes the Kd but not the Bmax of the radioligand.

Recipes

  1. Assay buffer
    50 mM TRIS: TRIS-HCl: 6.6 g and TRIS base 970 mg (or only 6.04 g TRIS base/L)
    5 mM KCl: 373 mg/L
    2 mM CaCl2: 220 mg/L
    2 mM MgCl2(6 H2O): 410 mg/L
    pH 7.4
  2. Wash buffer
    50 mM TRIS: TRIS-HCl (33 g/5 L) and TRIS base (4.85 g/5 L) (or only 30.22 g/5 L TRIS base)
    pH 7.4
  3. 0.1% PEI (only for D3 receptor assay)
    Prepare 0.1% solution, PEI is delivered as a 50% solution, pipet 1 ml from this with a syringe and add to 9 ml aqua dest. To get 5%, pipet 400 μl PEI 5% + 19.6 ml destilled water to get PEI 0.1%
  4. NSB solution
    3.98 mg butaclamol HCl/10 ml assay buffer yields 1.10-3 M (prepare aliquots of this)
    Dilute this 1:200 to obtain 5 μM in solution, i.e. 1 μM final concentration in assay
  5. Radioligand solutions
    The intended radioligand concentration range in the assay should be chosen to cover both the expected Kd and about 5-10x Kd (Kd spiperone in transfected cells according to literature ≈ 0.05 nM, 0.35 nM and 0.07 nM for D2, D3 and D4, respectively). The stock solution to be prepared needs to be 5x the assay concentration. Thus, the stock solution should be 25-50x Kd. The following is an example calculation based on a specific activity of 16.2 Ci/mmol radioactive of the radioactive stock solution for a D2 receptor assay. This needs to be adapted for the other subtypes based on their Kd values and for each batch of radioligand and its specific activity.
    [3H]-spiperone concentration = 1 mCi/ml/(16.2 Ci/mmol x 1,000 mCi/Ci) = 61.7 μM
    Highest concentration needed (10 x Kd x 5) = 10 x 0.05 x 5 = 2.5 nM
    2.5 ml x 2.5 nM = 0.00625 nmol [3H]-spiperone needed
    (0.00625 nmol)/61.7 x 103 nM = 0.1013 μl of [3H]-spiperone solution needed for 2.5 ml
    Thus 1 μl [3H]-spiperone solution in 25.0 ml assay buffer to yield a concentration of 2.5 nM.
    A free tool for such calculations can be found at www.graphpad.com/quickcalcs/chemMenu/.
  6. Radioligand Dilutions (Over a 100-fold Range) (Table 2).

    Table 2. Dilution scheme to for radioligand working solutions
    Number
    Relative concentration
    Preparation
    1
    100
    2.5 ml of radioligand at the highest concentration
    2
    66.67
    Mix 800 μl from tube 1 + 400 μl assay buffer
    3
    50
    Mix 1,000 μl from tube 1 + 1,000 μl assay buffer
    4
    25
    Mix 1,000 μl from tube 3 + 1,000 μl assay buffer
    5
    12.5
    Mix 1,000 μl from tube 4 + 1,000 μl assay buffer
    6
    6.25
    Mix 1,000 μl from tube 5 + 1,000 μl assay buffer

Acknowledgments

This protocol is the adaptation of a protocol originally published by Levant (2007).

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Levant, B. (2007). Characterization of Dopamine Receptors. Curr Protoc Pharmacol 36:1.6.1–1.6.15.
  3. van Wieringen, J. P., Booij, J., Shalgunov, V., Elsinga, P. and Michel, M. C. (2013). Agonist high- and low-affinity states of dopamine D2 receptors: methods of detection and clinical implications. Naunyn Schmiedebergs Arch Pharmacol 386(2): 135-154.


How to cite: Wieringen, J. v. and Michel, M. C. (2013). [3H]-Spiperone Saturation Binding to Dopamine D2, D3 and D4 Receptors. Bio-protocol 3(20): e945. DOI: 10.21769/BioProtoc.945; Full Text



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