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[3H]-Spiperone Competition Binding to Dopamine D2, D3 and D4 Receptors
[3H]-螺旋哌丁苯与多巴胺D2、D3和 D4受体的竞争性结合   

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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 competition experiment is usually performed to determine the affinity of a compound for a receptor. If multiple subtypes or states of the receptor are present and the competing compound differentiates them, a competition binding experiment can quantify the relative contribution of the two subtypes or states; while resolution of more than two subtypes or states is theoretically possible, in practical terms it is almost never feasible. Thus, radioligand binding to a receptor is quantified in the presence of various concentrations of the unlabelled compound of interest. The concentration of the radioligand in a competition study should be about 2-3 its Kd value as determined in saturation binding; this will allow a sufficient occupancy of the receptor to obtain a strong signal and at the same time avoid that competition becomes too difficult due to high radioligand concentration. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Of note, a simple competition experiments does not necessarily prove a competitive nature of the interaction between unlabelled drug and receptor. 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). The number of required competitor concentrations depends on the goal of the experiment. If only a rough estimate of antagonist potency is required, 1-2 concentration per log increment will be sufficient. However, if it is the aim to test for possible subtypes or states of the receptor, 3-5 concentrations per log increment are needed. If possible, the lowest competitor concentrations in the assays should not cause any detectable inhibition, whereas the highest concentrations should completely abolish specific binding. 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. Additionally, duplicates of total binding and non-specific binding should be included in the assay; 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(多巴胺), Receptor(受体), Radioligand(放射配体), Binding(结合), Spiperone(螺环哌啶酮)

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. Competitor compounds (dopamine HCl used as example here, e.g. Sigma-Aldrich, catalog number: 62-31-7 )
  5. Whatman GF/C filters (e.g. PerkinElmer, catalog number: 6005174 )
  6. Poly(ethyleneimine) solution (PEI) (e.g. Sigma-Aldrich, catalog number: 9002-98-6 )
  7. Scintillation cocktail (e.g. PerkinElmer, catalog number: 6013641 )
  8. TRIS base
  9. CaCl2
  10. MgCl2
  11. Distilled water
  12. Ascorbic Acid
  13. Assay buffer (see Recipes)
  14. Wash buffer (see Recipes)
  15. 0.1% PEI (only for D3 receptor assay) (see Recipes)
  16. NSB solution (see Recipes)
  17. Dilutions of competitors compound (see Recipes)
  18. Radioligand solution (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) or similar

Procedure

  1. Before the assay
    1. Prepare assay and wash buffer.
    2. For a D3 assay: 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 dilutions of competitor compound (dopamine used as example here).
    6. Prepare radioligand solution.
    7. Membrane preparation.
    8. 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 the desired protein concentration and to yield a total volume of about 8 ml per 48 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.
    9. Pre warm all solutions for 15 min in 25 °C waterbath.
    10. Final preparation (Table 1), add components to wells in following order:
      1. 250 μl assay buffer.
      2. 150 μl membrane suspension.
      3. 200 μl competitor, assay buffer (TB) or butaclamol (NSB). Use increasing concentrations of competitor.
      4. 200 μl assay buffer.
      5. Start reaction by adding 200 μl radioligand solution.

        Table 1. Pipetting scheme for competitor concentrations on microtiter plate
        TB
        TB
        NSB
        NSB
        10
        10
        2 x 10
        2 x 10
        3 x 10
        3 x 10
        5 x 10
        5 x 10
        9
        9
        2 x 9
        2 x 9
        3 x 9
        3 x 9
        5 x 9
        5 x 9
        8
        8
        2 x 8
        2 x 8
        3 x 8
        3 x 8
        5 x 8
        5 x 8
        7
        7
        2 x 7
        2 x 7
        3 x 7
        3 x 7
        5 x 7
        5 x 7
        6
        6
        2 x 6
        2 x 6
        3 x 6
        3 x 6
        5 x 6
        5 x 6
        5
        5
        NSB
        NSB














































        Total
        Total

  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 assay
    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 and 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 data being obtained can be analyzed to yield several types of information. The following should be considered.
    1. Carefully inspect raw data for consistency of replicates and resist the temptation to beautify the data by eliminating apparent ‘outliers’. It is our recommendation that one should very conservative in this regard; the result of a well-designed experiment will not be heavily affected by a single outlier. In other words, if the outcome of the experiment hinges on the question whether a single data point is an outlier, the overall experiment may have been designed and/or executed poorly and should probably be repeated.
    2. Plot amount of binding in the absence and presence of competitor on the y-axis vs. concentration of the competitor on the x-axis. In such plots the amount of binding in the absence of competitor (total binding, TB) can be entered at a virtual concentration lower than any tested competitor concentration, and non-specific binding (NSB) at a virtual concentration higher than the highest tested competitor concentration. A representative experiment with D2 receptors may look like this.



    3. Using all replicates without averaging them is preferable for the data analysis.
    4. In a well performed experiment, the concentration range of the competitor has been chosen in a way that the lowest competitor concentration does not cause any measurable inhibition and that the highest competitor concentration yields a degree of inhibition close to non-specific binding. If one of these two conditions is not met, data analysis becomes tricky as curve fitting may yield spurious values.
    5. Apply one of many available data analysis software packages, e.g. Prism, to fit a sigmoidal curve to the data. In most of such computer programs, you have a choice of settings which have important implications for the interpretation of the resulting parameter estimates.
      1. The preferred option is to let the top and the bottom of the curve to be found be the software, and the resulting values should be checked whether they are close to the experimentally determined total and non-specific binding.
      2. The turning point of the sigmoidal curve should be found by the software in most cases as this will yield the IC50 of the curve.
      3. The slope of the curve can be found by the software. Values close to unity indicate interaction with a single site. If the slope is smaller than unity, i.e. the curve is shallow, this may indicate interaction with multiple sites (see below) but does not necessarily prove it.
      4. If the assumption is made that an interaction with multiple sites, e.g. receptor subtypes, is possible, it can be defined that the curves should follow a monophasic or a biphasic function with each component having a slope of unity. On theoretical grounds a biphasic fit always yields a smaller residual error, as there are more fitting parameters. Therefore, a biphasic fit should only be accepted if it yields a significantly smaller residual error as judged by an F-test or similar (already implemented in most software packages for such use). If a biphasic fits is superior, it will yield IC50 values for both components and a percentage of each of the two components. e.g. for the representative experiment shown above, the sum of squares of distances of data points to the fitted curve was 24177 and 18221 for the one- and two-site fit, respectively, yielding an F value of 6.734, which indicates with a p-value of 0.0040 that the null hypothesis (single site is preferred) should be rejected and a two-site model should be preferred.
    6. The ability to pick up biphasic curves depends on the selectivity of the competitor for the two subtypes being present (or the intrinsic activity of the agonist, if agonist high- and low-affinity states are being analyzed) as well as the number of inhibitor concentrations being tested per log unit increment of concentration.
    7. The results of the curve fitting should always be inspected visually to check whether they make sense. e.g. in some cases the computer program yields estimates which are outside the range of tested competitor concentrations. Such extrapolations are highly unreliable and should not be used; rather the experiment should be repeated with a better choice of competitor concentrations.
    8. Estimates of IC50 depend on the concentration of radioligand in the assay, expressed as fold of its Kd value. To obtain the more informative Ki value, transformation of IC50 is necessary by the Cheng & Prusoff equation:
      Ki = IC50/((L/Kd) + 1)
      in which L and Kd are the concentration and affinity of the radioligand.
      Notes:
      1. A competition binding experiment by virtue of its design cannot prove a competitive interaction between inhibitor and radioligand. This requires e.g. saturation experiments in the absence and presence of one or more inhibitor concentrations.
      2. As IC50 values are obtained from a log scale, the replicates from multiple experiments typically do not exhibit a Gaussian distribution on a linear scale. Hence, the average from multiple experiments should be presented as means (with SD) of –log IC50 (or –log Ki). Alternatively, the median IC50 or Ki can be presented with (asymetric) confidence intervals. Means (with SD) of linear IC50 or Ki values are inappropriate.

Recipes

  1. Assay buffer
    50 mM TRIS: TRIS-HCl: 6.6 g and TRIS base 970 mg/L (or only 6.04 g TRIS base/L)
    5 mM KCl: 373 mg/L
    2 mM CaCl2:  220 mg/L
    2 mM MgCl2.6H2O: 410 mg/L
    pH 7.4
    Every test day prepare a fresh buffer containing 0.05% (25 mg/50 ml) ascorbic acid
    Note: This buffer is optimized for detection of agonist high-affinity states of a receptor (agonist competition curves); hence, inclusion of Na+ should be avoided. If that is not within the scope of the project, a buffer without Mg2+, Ca2+ or K+ can be used.
  2. Wash buffer
    50 mM TRIS: TRIS-HCl (33 g/5 L) and TRIS base (4.85 g/5 L)
    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 asay
  5. Dilutions of competitors compound (dopamine used as example here)
    1. Molecular weight of dopamine HCl is 189.6 g/mol. Weigh 3-5 mg, calculate needed volume of assay buffer to reach target concentration; trying to start with a fixed volume and to reach a specific amount of compound is technically more difficult. In this example 3.8 mg has to be dissolved in 20 ml assay buffer to reach 10-3 M. Dilute this 2x with assay buffer to reach 5x the intended highest concentration in the assay, e.g. 5 x 10-4 M for 10-4 M in the assay.
    2. In the first row of below scheme place 1,800 μl assay buffer per tube, in the 2nd row (2x) 800 μl, in the 3rd row (3x) 700 μl and in the 4th row (5x) 500 μl. Amounts to be prepared may need to be increased if more than one competition experiment is to be performed.
    3. Dilute competitor stock solution in 1:10 steps first. Pipet from 10-4 200 μl to 5 and from 5,200 μl to 6 etc. Then, make 2x, 3x and 5x daughter solutions, pipet 200 μl in 2x, 300 μl in 3x and 500 μl in 5x. Do this also for 8, 7, 6, 5 and 4. (Table 2)

      Table 2. Dilution scheme for competitor working solutions
      5x   
       5x   
       5x   
      5x   
        5x  





      NSB
      3x   
      3x  
      3x  
      3x   
      3x







        2x     
       2x   
         2x     
      2x   
      2x







      9    
      8   
      7   
      6   

      4
        
        
        
        
        


  6. Radioligand solution
    The intended radioligand concentration in the assay should be about 2-3x 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 10-15x 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. Prepare about 10 ml of stock solution per planned 48 data point experiment. Stock solution can be prepared in 50 ml tube with screw cap.
    [3H]-spiperone concentration = 1 mCi/ml/(16.2 Ci/mmol x 1,000 mCi/Ci) = 61.7 μM
    Highest concentration needed (3 x Kd x 5) = 3 x 0.05 x 5 = 0.75 nM
    10 ml x 0.75 nM = 0.0075 nmol [3H]-spiperone needed
    (0.0075 nmol)/61.7 x 103 nM = 0.12 μl of [3H]-spiperone solution needed for 10 ml
    Thus 1 μl [3H]-spiperone solution in 83.3 ml assay buffer to give a concentration of 0.75 nM
    A free tool for such calculations can be found at www.graphpad.com/quickcalcs/chemMenu/.

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 D(2) receptors: methods of detection and clinical implications. Naunyn Schmiedebergs Arch Pharmacol 386(2): 135-154.

简介

该方案旨在用于96孔板(1,200μl孔),但它可以类似地应用于标准试管(Levant,2007)。 D 2,D 3和D 4多巴胺受体是D 2 S 2类多巴胺受体的成员受体。可以使用放射性配体[3 H] - 螺哌隆来研究它们,所述放射性配体是结合D 2,D 3和D 3的拮抗剂, 4>受体。通常进行竞争实验以确定化合物对受体的亲和力。如果存在受体的多种亚型或状态,并且竞争性化合物使它们分化,则竞争结合实验可以量化两种亚型或状态的相对贡献;而在两个以上亚型或状态的分辨率在理论上是可能的,在实际中它几乎是不可行的。因此,在各种浓度的未标记的目的化合物的存在下,定量放射性配体与受体的结合。在竞争研究中放射性配体的浓度应当是在饱和结合中测定的约2-3的K d值;这将允许受体的充分占据以获得强信号,并且同时避免由于高放射性配体浓度而使竞争变得太难。选择孵育时间和温度以允许与放射性配体和竞争剂的受体的缔合和解离之间形成平衡。值得注意的是,简单的竞争实验不一定证明未标记的药物和受体之间的相互作用的竞争性。如果相对于放射性配体的亲和力(<1nM),比放射性低(氚化),则需要高测定体积(≥1ml)以避免配体消耗;如果使用具有高表达密度的受体来源(例如表达的重组受体),这是特别重要的。所需竞争剂浓度的数量取决于实验的目标。如果仅需要对拮抗剂效力的粗略估计,则每个对数增量1-2个浓度就足够了。然而,如果目的是测试受体的可能亚型或状态,则需要每个对数增量3-5个浓度。如果可能,测定中最低的竞争剂浓度不应引起任何可检测的抑制,而最高浓度应完全消除特异性结合。每个实验可以分为不同的步骤,如测定制备,膜制备,温育,过滤,样品计数和数据分析。为了使实验误差最小化,所有测定至少一式两份进行。另外,总结合和非特异性结合的重复应包括在测定中;用于定义非特异性结合(NSB)的试剂应当是与放射性配体不同的化学(不同的家族)和物理上(避免两种亲脂性化合物的组合),以避免伪像。对于所选择的测定条件的具体益处的讨论参见van Wieringen等人(2013)(拷贝可以从作者获得)。

关键字:多巴胺, 受体, 放射配体, 结合, 螺环哌啶酮

材料和试剂

  1. 放射性配体[3 H] - 螺哌隆(例如PerkinElmer,目录号:NET565250UC)
  2. Butaclamol(如 Sigma-Aldrich,目录号:55528-07-9)
  3. 含受体的膜悬浮液
  4. 竞争性化合物(这里使用的多巴胺HCl,例如Sigma-Aldrich,目录号:62-31-7)
  5. Whatman GF/C过滤器(例如 PerkinElmer,目录号:6005174)
  6. 聚(乙烯亚胺)溶液(PEI)(例如Sigma-Aldrich,目录号:9002-98-6)
  7. 闪烁鸡尾酒(例如:PerkinElmer,目录号:6013641)
  8. TRIS基地
  9. CaCl <2>
  10. MgCl 2
  11. 蒸馏水
  12. 抗坏血酸
  13. 测试缓冲区(参见配方)
  14. 洗涤缓冲液(见配方)
  15. 0.1%PEI(仅用于D 3受体测定)(参见配方)
  16. NSB解决方案(参见配方)
  17. 稀释的竞争对手复合(见配方)
  18. 放射性配体溶液(参见配方)

设备

  1. 96孔板(聚苯乙烯)
  2. 细胞收集器(例如 PerkinElmer)
  3. Ultra-Turrax(IKA,型号:0001602800)或类似的分散器
  4. 水浴
  5. 闪烁计数器

软件

  1. Prism(Graphpad Software)或类似软件

程序

  1. 测定前
    1. 准备测定和洗涤缓冲液。
    2. 对于D 3测定:在滤板上制备0.1%PEI溶液和移液管100μl/过滤器,随后置于冰箱(4℃)中至少2小时。
    3. 在总测定体积为1,000μl(450μl测定缓冲液+200μl放射性配体+200μl测定缓冲液或5μM(+) - butaclamol +150μl膜制备物)的96孔板(聚苯乙烯)中进行实验。
    4. 准备NSB解决方案。
    5. 制备竞争性化合物(此处使用多巴胺)的稀释液。
    6. 准备放射性配体溶液
    7. 膜制备。
    8. 根据制备方案制备含受体的膜悬浮液。使用Ultra-Turrax短脉冲使小体积(<2ml)中的悬浮液再匀化。稀释至所需的蛋白质浓度,并且每48个数据点实验产生约8ml的总体积。应该选择膜悬浮液的蛋白质浓度,以便获得稳定的特异性结合信号,但同时, 10%(甚至更好地<5%)的游离放射性配体浓度。蛋白质含量可以通过多种文章来测定,例如Br​​adford(1976)。首先在冰中制备膜悬浮液。
    9. 预温所有解决方案,在25°C水浴中15分钟。
    10. 最后准备(表1),按以下顺序向孔中添加组分:
      1. 250μl测定缓冲液
      2. 150μl膜悬浮液
      3. 200μl竞争剂,测定缓冲液(TB)或butaclamol(NSB)。 使用越来越浓的竞争对手。
      4. 200μl测定缓冲液
      5. 通过加入200μl放射性配体溶液开始反应。

        表1.微量滴定板上竞争剂浓度的移液方案
        TB
        TB
        NSB
        NSB
        10
        10
        2 x 10
        2 x 10
        3 x 10
        3 x 10
        5 x 10
        5 x 10
        9
        9
        2 x 9
        2 x 9
        3 x 9
        3 x 9
        5 x 9
        5 x 9
        8
        8
        2 x 8
        2 x 8
        3 x 8
        3 x 8
        5 x 8
        5 x 8
        7
        7
        2 x 7
        2 x 7
        3 x 7
        3 x 7
        5 x 7
        5 x 7
        6
        6
        2 x 6
        2 x 6
        3 x 6
        3 x 6
        5 x 6
        5 x 6
        5
        5
        NSB
        NSB














































        总计
        总计

  2. 在测定期间
    1. 在25℃水浴中孵育120分钟
    2. 使用细胞收集器通过Whatman GF/C过滤器快速真空过滤终止反应。 用冰冷的洗涤缓冲液洗涤过滤器10次
  3. 测定后
    1. 将50μl放射性配体的等分试样加入计数板的孔中以测定总放射性
    2. 干燥,例如。 在烤箱中烤2小时。
    3. 将贴纸放在底部,加入闪烁鸡尾酒(20μl)至过滤器,将贴纸置于板的顶部,并在闪烁计数器中计数,在计数样品前允许足够的时间(15分钟)。
  4. 数据分析
    可以分析所获得的数据以产生几种类型的信息。应考虑以下内容。
    1. 仔细检查原始数据的重复一致性,并通过消除明显的"异常值"抵制美化数据的诱惑。我们建议在这方面应该非常保守;设计良好的实验的结果不会受到单个离群值的严重影响。换句话说,如果实验的结果取决于单个数据点是否是异常值的问题,则整个实验可能已被设计和/或执行得不好,并且应该重复。
    2. 在y轴上竞争剂不存在和存在下的结合量与竞争剂在x轴上的浓度的图。在这些图中,可以在低于任何测试的竞争剂浓度的虚拟浓度下进入在不存在竞争剂(总结合,TB)的情况下的结合量,以及在高于最高测试竞争剂的虚拟浓度下的非特异性结合(NSB)浓度。具有D 2受体的代表性实验可能看起来像这样


    3. 对于数据分析,使用所有重复,而不进行平均是更可取的。
    4. 在良好实施的实验中,竞争剂的浓度范围以如下方式选择:最低竞争剂浓度不引起任何可测量的抑制,并且最高竞争剂浓度产生接近非特异性结合的抑制程度。如果不满足这两个条件之一,则数据分析变得棘手,因为曲线拟合可能产生假值
    5. 应用许多可用的数据分析软件包之一,例如

      Prism,以便为数据拟合S形曲线。在大多数这样的计算机程序中,您可以选择对解释结果参数估计有重要影响的设置。
      1. 首选方法是让曲线的顶部和底部作为软件,并检查结果值是否接近实验确定的总和非特异性绑定。
      2. 在大多数情况下,软件应该找到S形曲线的转折点,因为这将产生曲线的IC 50
      3. 曲线的斜率可以通过软件找到。接近1的值表示与单个站点的相互作用。如果斜率小于单位,则曲线是浅的,这可以指示与多个站点的交互(见下文),但不一定证明它。
      4. 如果假设与多个位点例如受体亚型的相互作用是可能的,则可以定义为曲线应该遵循单相或双相函数,每个分量具有单位斜率。在理论上,双相拟合总是产生更小的残余误差,因为存在更多的拟合参数。因此,如果通过F检验或类似的(已经在用于这种用途的大多数软件包中实现的)判断,产生显着更小的残留误差,则应当接受双相拟合。如果两相拟合优越,则它将产生两个组分的IC 50值和两个组分中的每一个的百分比。例如对于上面所示的代表性实验,数据点到拟合曲线的距离的平方和分别为24177和18221,对于单位点和两位点拟合,产生6.734的F值,其以p值0.0040指示应该拒绝零假设(优选单个位点),并且优选双位点模型。
    6. 拾取双相曲线的能力取决于竞争剂对存在的两种亚型的选择性(或者如果激动剂高和低亲和力状态正在被分析,则是激动剂的固有活性)以及抑制剂浓度的数量每个浓度单位增量测试
    7. 曲线拟合的结果应该总是被目视检查以检查它们是否有意义。例如,在一些情况下,计算机程序产生超出测试的竞争者浓度范围的估计。这种外推是非常不可靠的,不应该使用;而是应该重复实验,更好地选择竞争对手的浓度
    8. IC 50的估计值取决于测定中放射性配体的浓度,表示为其K d值的倍数。为了获得更多信息的Ki值,IC 50的转化是必需的, Prusoff方程:
      K sub = IC 50 /((L/K sub)+ 1)
      其中L和K d是放射性配体的浓度和亲和力 注意:
      1. 由于其设计的竞争结合实验不能证明抑制剂和放射性配体之间的竞争性相互作用。这需要例如。在一种或多种抑制剂浓度不存在和存在下的饱和实验。
      2. 从对数标度获得IC 50值,来自多个实验的重复通常不在线性标度上呈现高斯分布。 因此,来自多个实验的平均值应该表示为-log IC 50(或-log K sub)的平均值(具有SD)。 或者,可以用(不对称的)置信区间来呈现中值IC 50或K i。 线性IC 50 或K i 值的均值(与SD)不合适。

食谱

  1. 测定缓冲区
    50mM TRIS:TRIS-HCl:6.6g和TRIS碱970mg/L(或仅6.04g TRIS碱/L)
    5mM KCl:373mg/L
    2mM CaCl 2 :< 220 mg/L
    2mM MgCl 2 6H 2 O:410mg/L
    pH 7.4
    每个测试日制备含有0.05%(25mg/50ml)抗坏血酸的新鲜缓冲液 注意:该缓冲液优化用于检测受体的激动剂高亲和力状态(激动剂竞争曲线);因此,应避免包括Na + 。如果这不在项目的范围内,则没有Mg 2 + ,Ca 2 + 或K +
  2. 洗涤缓冲液
    50mM TRIS:TRIS-HCl(33g/5L)和TRIS碱(4.85g/5L)
    pH 7.4
  3. 0.1%PEI(仅用于D 3受体测定) 制备0.1%溶液,PEI作为50%溶液递送,用注射器从其中移取1ml,并加入9ml水。得到5%,移取400μlPEI 5%+ 19.6ml蒸馏水得到PEI 0.1%
  4. NSB解决方案
    3.98mg丁草胺HCl/10ml测定缓冲液产生1μL。 10 M(制备其等分试样)
    稀释该1:200以获得5μM的溶液,即在终浓度为1μM的最终浓度。
  5. 竞争化合物的稀释液(多巴胺用作此处的实例)
    1. 多巴胺HCl的分子量为189.6g/mol。 称重3-5 mg, 计算测定缓冲液达到目标浓度所需的体积; 尝试从固定的卷开始并达到特定量 复合在技术上更困难。 在这个例子中,必须是3.8mg   溶解在20ml测定缓冲液中以达到10μM-3μM。稀释2倍 测定缓冲液达到预期最高浓度的5倍 测定中,例如对于10μM-4μM的5×10 4-4μM。
    2. 在里面 第一行以下方案每管放置1,800μl测定缓冲液 在第3行(3x)700μl和第4行(5x)中的第2行(2x)800μl中, 500μl。 如果超过一个,可能需要增加准备的金额   将进行竞争实验。
    3. 稀 竞争对手库存溶液以1:10步骤首先。 吸取从10 -4 200μl至   5和5,200μl至6μl等。 然后,做2x,3x和5x的女儿 溶液,移液管中200μl在2x,300μl在3x和500μl在5x。 做这个 也为8,7,6,5和4.(表2)

      表2.竞争对手工作解决方案的稀释计划
      5x   
        5x   
        5x   
      5x   
        5x  





      NSB
      3x   
      3x  
      3x  
      3x   
      3x







        2x     
        2x   
         2x     
      2x   
      2x







      9    
      8   
      7   
      6   

      4
        
        
        
        
        


  6. 放射性配体溶液
    预期的放射性配体浓度 测定法在转染细胞中应当是约2-3×K d(K d subspiperone) 根据对于D 2,D 3和D 3的文献≈0.05nM,0.35nM和0.07nM, D <4>)。准备的储备溶液需要是5倍 测定浓度。因此,储备溶液应当为10-15×K d。的 以下是基于16.2的特定活动的示例计算  Ci/mmol放射性D 2受体的放射性储备溶液  测定。这需要适应基于他们的其他亚型 K d值和每批放射性配体及其比活性。 每个计划的48个数据点准备约10ml的储备溶液 实验。储备溶液可以在带有螺帽的50ml管中制备 [3 H] - 螺哌隆浓度= 1mCi/ml /(16.2Ci/mmol×1,000mCi/Ci)=61.7μM
    所需的最高浓度(3×K d×5)= 3×0.05×5 = 0.75nM
    10ml×0.75nM = 0.0075nmol [3 H] - 螺环酮需要
    (0.0075nmol)/61.7×103nM=0.12μl10ml所需的[3 H] - 螺环哌啶酮溶液
    因此,在83.3ml测定缓冲液中的1μl[3 H] - 螺哌隆溶液,得到浓度为0.75nM的
    有关此类计算的免费工具,请访问 www.graphpad.com/quickcalcs/chemMenu/.

致谢

该协议是Levant最初发布的协议(2007)的改编。

参考文献

  1. Bradford,MM(1976)。一种快速灵敏的微量蛋白定量定量方法 蛋白染料结合的原理。 Anal Biochem 72:248-254
  2. Levant,B。(2007)。 多巴胺受体的表征。 36:1.6.1-1.6.15。
  3. van Wieringen,J.P.,Booij,J.,Shalgunov,V.,Elsinga,P.and Michel,M.C。(2013)。 多巴胺D(2)受体的激动剂高和低亲和力状态:检测和临床的方法 意义。 Naunyn Schmiedebergs Arch Pharmacol 386(2):135-154。
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引用:Wieringen, J. v. and Michel, M. C. (2013). [3H]-Spiperone Competition Binding to Dopamine D2, D3 and D4 Receptors. Bio-protocol 3(20): e944. DOI: 10.21769/BioProtoc.944.
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