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Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
基于位移的酶联免疫吸附试验:配体竞争结合Ni2+-NTA板上His标签能力的定量分析   

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Abstract

The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn2+ or with EDTA to chelate Zn2+) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni2+-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was quantified with antibodies specific to the S18-2 protein and secondary antibodies, in chemiluminescent ELISA. In this way displacement of S18-2 protein by the S18-1 protein was reported as a percentage of the full strength signal achieved through saturation of S6 with S18-2. At its foundation, this method exploits a native protein-protein interaction and could be applied to other systems where two or more proteins compete for binding to a target ligand as above.

Keywords: ELISA(ELISA), His tag(他的标签), Protein-protein interaction(蛋白质-蛋白质相互作用)

Materials and Reagents

  1. HisPurTM Cobalt Spin Columns (Thermo Fisher Scientific, catalog number: 89969 )
  2. Pierce™ Polyacrylamide Spin Desalting Columns, 7 K MWCO, 0.7 ml (Thermo Fisher Scientific, catalog number: 89849 )
  3. 5 PRIMETM Ni2+-NTA HisPrimeTM plates (Thermo Fisher Scientific, catalog number: 2400730 )
  4. ProTEV Plus (Promega Corporation, catalog number: V6101 )
  5. cOmplete His-Tag Purification Resin (Sigma-Aldrich, Roche Diagnostics, catalog number: 05893682001 )
  6. BioRad Protein Concentration Assay (Bio-Rad Laboratories, catalog number: 5000002 )
  7. Albumin, Bovine, Fraction V (pH 7) (BSA) (Affymetrix, catalog number: 10857 50 gm )
  8. Rabbit Polyclonal Antibody raised to peptide antigen from competing protein (i.e. S18-2) (NeoPeptide, custom made against peptide PGQDRQRRAALCP)
  9. Goat Anti-Rabbit IgG, Peroxidase Conjugated Secondary Antibody (Thermo Fisher Scientific, PierceTM, catalog number: 31460 )
  10. SuperSignalTM West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34080 )
  11. HEPES Sodium Salt (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP410-500 )
  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 793590-500 g )
  13. Tween® 20 (enzyme grade) (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP337-500 )
  14. 2-mercaptoethanol (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP176-100 )
  15. EDTA disodium salt (Thermo Fisher Scientific, Fisher BioReagents, catalog number: BP120-500 )
  16. ZnSO4.7H2O (Sigma-Aldrich, catalog number: Z4750-100 g )
  17. 5x HEPES/KCl (see Recipes)
  18. Microscale Thermophoresis Buffer (MST) buffer (see Recipes)
  19. MST with Zn2+ (see Recipes)
  20. MST with EDTA (see Recipes)

Equipment

  1. Multichannel pipettor (20-300 µl) (Gilson, model: FA10016 )
  2. Microplate reader with absorbance and chemiluminescent capacity (BioTek Instruments, model: Synergy 2 )
  3. Benchtop microplate shaker (IKA® Works, model: MS 3 digital )

Procedure

In this experiment, we have defined the His-tagged cognate binding partner as the immobilized ligand (S6-His) that interacts with two competing proteins, which are not immobilized (S18-1 or S18-2). Either of the two competing binding partners could be used to displace the other. However, we had antibodies only against the S18-2 protein and therefore the displacement protocol described here allows for detection of the S18-2 protein remaining bound to the S6 protein after displacement with the S18-1 protein. In general, if proteins binds to the same binding site with similar on and off rates, the order of addition of the competing proteins should not matter. Before proceeding with the experiment, there are two controls that need to be established. First, each competing protein should be added to separate wells without the presence of the His-tagged cognate protein. We did this control to confirm complete removal of the His-tag and to confirm the competing protein did not bind to the plate without the presence of S6-His. The second control is to determine the molar amount of the His-tagged protein that gives a linear range of detection, before the Ni2+-NTA binding sites become saturated. The plates used in this assay are 96 well Ni2+-NTA coated plates with a reported binding capacity of 10-20 pmol/well. We tested a range of His-tagged protein amounts from 5-150 pmol/well and found that the signal was saturated at 80 pmol when using 200 µl volume per well. Therefore, we used less than 80 pmol/well of the S6-His protein for the assay to ensure the molar concentrations of the competing untagged S18 proteins would be in excess of the His-tagged protein bound to the plate. Always use freshly purified and refolded proteins (or thawed aliquots stored at -80 °C) and ice-cold buffer for all dilutions and washes to maintain integrity of the ribosomal proteins throughout the assay.

  1. To begin this experiment purified recombinant proteins are needed. If His-tag is used for purification, the competing proteins, as in case of S18-1 and S18-2, must have their tags removed while the cognate protein/ligand, i.e. S6 protein, must remain His-tagged. As described previously (Prisic et al., 2015), we used hexa-histidine tags for S18-1, S18-2, and S6 and purified the recombinant proteins on cobalt columns under denaturing conditions and then refolded by dialysis. Using an engineered TEV cleavage site, we removed the histidine tag from S18-1 and S18-2 with TEV protease, followed by purification of the cleaved proteins with a His-tag purification resin matrix.
  2. Ensure the proteins are all in the same buffer; in this case MST buffer was used. If they are not in the same buffer, they should be re-dialyzed or buffer should be exchanged using gel filtration. The ribosomal proteins were refolded and stored in MST buffer, which was optimized for binding studies using microscale electrophoresis (Prisic et al., 2015). Considering that we observed strong interaction between S6 and each S18 protein in this buffer, we decided to use the same conditions in the displacement assay. Components did not interfere with Ni2+-NTA-His tag binding and had acceptable effect on ELISA, i.e. binding of primary and secondary antibodies, and HRP activity. MST buffer was used for all dilutions, including antibody dilutions, as well as washing steps. We did this assay in parallel, one with proteins dialyzed with Zn2+ and one with proteins dialyzed with EDTA. Therefore we would use MST with Zn2+ (10 µM ZnSO4) when working with Zn2+-dialyzed proteins or MST with EDTA (100 µM EDTA) when working with EDTA-dialyzed proteins.
  3. Determine the concentration of the proteins. For the ribosomal proteins, we used BioRad protein concentration assay, a modified Bradford method. BSA standards were prepared in MST buffer and protein concentration was determined from the standard curve when measuring the absorbance at 595 nm in a microplate reader. Molecular weights of the proteins were 11.9 kDa for S6-His, 9.6 kDa for S18-1 and 9.8 kDa for S18-2. The molar concentration of the proteins was determined from the protein concentration given from the Bradford assay using the following formula:

  4. Make a master mix of His-tagged protein diluted in MST for all wells to be tested. We used 30 pmol/well of the His-tagged protein and 200 µl well volumes, so the protein will be diluted to a final concentration of 150 µM. Aliquot 200 µl of diluted His-tagged protein into each well (Figure 1a). It is recommended that at least two to three technical replicates are done in parallel.
  5. Incubate plate for 30 min at room temperature with shaking at 300 rpm on a bench top plate shaker.
  6. Discard the solution from the plate and wash the wells four times with 200 µl MST. Washes are done with ice-cold buffer but are carried out at room temperature. Wells are soaked with wash buffer for 30-60 sec without shaking and tapped dry onto paper towels between each wash.
  7. Make a master mix of the S6 binding partner (i.e. S18-2 protein) at a molar concentration at least 1.5 times greater than that used for the His-tagged protein, so there is enough material to ensure complete saturation of the His-tagged protein. In our case, we used a molar concentration of 270 µM (54 pmol/well) of the S18-2 protein. Aliquot 200 µl of the master mix per well (Figure 1b).
  8. Incubate plate for 30 min at room temperature with shaking (300 rpm) on a bench top plate shaker.
  9. Discard the solution from the plate and wash as done in step 6.
  10. Prepare a range of molar concentrations for another S6-binding protein (i.e. S18-1) in the following manner. Prepare a solution at 4x the amount of the first S6 binding partner used in step 7 (in our case this is 1.08 mM). Create serial dilutions decreasing the concentration by half each step until 0.25x is achieved (67.5 µM).
  11. Aliquot 200 µl of each dilution per well. Include one well to which no competing protein is added to establish the full strength signal obtained from saturation of the His-tagged cognate protein without displacement (Figure 1c).
  12. Incubate plate for 30 min at room temperature with shaking (300 rpm) on a bench top plate shaker.
  13. Discard the solution from the plate and wash as done in step 6.
  14. Prepare the primary antibody (antibody specific to one of the competing proteins, in our case it was against S18-2) at 1:1,000 dilution in MST with 3% BSA. Aliquot 200 µl of the antibody solution to each well.
  15. Incubate plate for 30 min at room temperature with shaking (300 rpm) on a bench top plate shaker.
  16. Discard the solution from the plate and wash as done in step 6.
  17. Prepare the secondary antibody (conjugated to HRP) at 1:5,000 dilution in MST with 3% BSA. Aliquot 200 µl of the secondary antibody solution into each well, including controls.
  18. Incubate plate for 30 min at room temperature with shaking (300 rpm) on a bench top plate shaker.
  19. Discard the solution from the plate and wash as done in step 6.
  20. Using a multichannel pipettor, aliquot 200 µl of chemiluminescent substrate (e.g. 1:1 mix of SuperSignal Pico stable peroxide and substrate) to each well. Attempt to add substrate to each row of the plate (in our case each row was one complete replicate) in rapid succession, as the signal increases rapidly with time. Using a trough with premixed substrate will allow minimal rounds of pipetting. It is most important that each row of the plate has substrate added simultaneously using the multichannel pipette so that standardization of the well without competing protein and the treatment wells with competition for each replicate have substrate added simultaneously.
  21. Gently shake the plate for 1 min and read chemiluminescent signal (LUM) in a plate reader immediately (Figure 1d).
  22. Normalize the signal for each well to the signal obtained from the well in which no competing protein was added, representing a fully saturated signal. The signal obtained from each treatment well will be expressed as a percentage of the full strength signal obtained without competition (Figure 1e).

Representative data


Figure 1. Stepwise depiction of a displacement-based ELISA. Virtual cross-sections through six wells of a 96-well plate are depicted throughout stages of the displacement assay. a). The ligand (L) is immobilized on a Ni2+-NTA plate via His-tag. b). The first binding partner, P1, is added in excess to saturate the binding sites offered by the immobilized ligand. c). The second binding partner, P2, is added to the wells in increasing amounts from 0-4X of the maximal theoretical concentration of P1 bound to L. In the event that displacement of P1 by P2 occurs, some of the P1 that was originally bound to L will be replaced with P2. The amount of P1 that is displaced will be dependent on the ratio of P2/P1 that was added to the well, with higher ratios leading to less P1 that remains bound with L. d). The result of the displacement assay is obtained through an ELISA with primary antibodies specific to P1 and secondary antibodies conjugated to horseradish peroxidase (HRP). Light emitted from a luminescent HRP substrate is P1 concentration-dependent and displacement is determined through normalization of the signal from each well to one in which no P2 was introduced (i.e. full saturation of L with P1). e). Displacement can be visualized by plotting the normalized signal of P1 across the tested range of P2/P1 ratios. The graph shows two hypothetical results where displacement or no displacement occurred. In the scenario described in the protocol P1 is S18-2, P2 is S18-1 and L is S6-His.

Recipes

  1. 5x HEPES/KCl (1 L)
    123 g KCl
    23.83 g HEPES Sodium Salt
    900 ml H2O
    Adjust pH to 7.6 with NaOH
    Bring final volume to 1 L
  2. MST buffer (20 mM HEPES pH 7.6, 330 mM KCl, 0.05% Tween 20, 14 mM 2-mercaptoethanol) (1 L)
    200 ml 5x HEPES/KCl
    2.5 ml 20% Tween® 20
    1 ml 2-mercaptoethanol
    Bring final volume to 1 L
    Note: Always make MST buffer fresh and use the same day.
  3. MST with Zn2+
    MST buffer + 10 µM ZnSO4
  4. MST with EDTA
    MST buffer + 100 µM EDTA

Acknowledgments

This work is supported by startup funds from University of Hawaii at Manoa.

References

  1. Prisic, S., Hwang, H., Dow, A., Barnaby, O., Pan, T. S., Lonzanida, J. A., Chazin, W. J., Steen, H. and Husson, R. N. (2015). Zinc regulates a switch between primary and alternative S18 ribosomal proteins in Mycobacterium tuberculosis. Mol Microbiol 97(2): 263-280.

简介

设计置换测定以定量来自结核分枝杆菌(Mycobacterium tuberculosis)S18-1和S18-2的两个同源核糖体蛋白质之间的直接竞争,用于与它们的同源结合伴侣,核糖体蛋白S6(Prisic et al。,2015)。 S18蛋白质在两种生理相关条件下(即在Zn 2+ 2+存在下或用EDTA螯合Zn 2+ 2+)进行透析,并且然后允许竞争结合S6,其维持在有限浓度。通过ELISA获得结果,其中S6-His首先结合Ni 2+ 2+ -NTA板,然后加入超过S6的S18-2,然后加入递增浓度的S18-1。在化学发光ELISA中用S18-2蛋白和第二抗体特异性的抗体定量保留结合S6的S18-2的百分比。以这种方式,S18-1蛋白质被S18-1蛋白质的置换报告为通过用S18-2饱和S6实现的全强度信号的百分比。在其基础上,该方法利用天然蛋白质 - 蛋白质相互作用,并且可以应用于其中两种或更多种蛋白质竞争结合上述靶配体的其他系统。

关键字:ELISA, 他的标签, 蛋白质-蛋白质相互作用

材料和试剂

  1. HisPur TM Cobalt Spin Columns(Thermo Fisher Scientific,目录号:89969)
  2. Pierce TM聚丙烯酰胺旋转脱盐柱,7K MWCO,0.7ml(Thermo Fisher Scientific,目录号:89849)
  3. 5 PRIME TM sup/Ni Ni 2+ -NTA HisPrime TM板(Thermo Fisher Scientific,目录号:2400730)
  4. ProTEV Plus(Promega Corporation,目录号:V6101)
  5. cOmplete His-Tag纯化树脂(Sigma-Aldrich,Roche Diagnostics,目录号:05893682001)
  6. BioRad蛋白浓缩测定(Bio-Rad Laboratories,目录号:5000002)
  7. 白蛋白,牛,级分V(pH7)(BSA)(Affymetrix,目录号:1085750gm)
  8. 从竞争蛋白(即S18-2)(NeoPeptide,针对肽PGQDRQRRAALCP定制)对肽抗原产生的兔多克隆抗体
  9. 山羊抗兔IgG,过氧化物酶缀合的第二抗体(Thermo Fisher Scientific,Pierce TM ,目录号:31460)
  10. SuperSignal TM West Pico Chemiluminescent Substrate(Thermo Fisher Scientific,目录号:34080)
  11. HEPES钠盐(Thermo Fisher Scientific,Fisher BioReagents,目录号:BP410-500)
  12. 氯化钾(KCl)(Sigma-Aldrich,目录号:793590-500g)
  13. Tween 20(酶级)(Thermo Fisher Scientific,Fisher BioReagents,目录号:BP337-500)
  14. 2-巯基乙醇(Thermo Fisher Scientific,Fisher BioReagents,目录号:BP176-100)
  15. EDTA二钠盐(Thermo Fisher Scientific,Fisher BioReagents,目录号:BP120-500)
  16. ZnSO 4·7H 2 O(Sigma-Aldrich,目录号:Z4750-100g)
  17. 5x HEPES/KCl(参见配方)
  18. 微量热泳缓冲液(MST)缓冲液(参见配方)
  19. MST与Zn 2 + (参见配方)
  20. 含有EDTA的MST(参见配方)

设备

  1. 多通道移液器(20-300μl)(Gilson,型号:FA10016)
  2. 具有吸光度和化学发光能力的微板读数器(BioTek Instruments,型号:Synergy 2)
  3. 台式微量培养板振荡器(IKA Works,型号:MS 3数字)

程序

在这个实验中,我们定义了His标记的同源结合配偶体作为与两个竞争蛋白(S18-1或S18-2)相互作用的固定配体(S6-His)。两个竞争性结合配偶体中的任一个可以用于替换另一个。然而,我们具有仅针对S18-2蛋白的抗体,因此本文描述的置换方案允许在用S18-1蛋白置换后检测保留结合于S6蛋白的S18-2蛋白。一般来说,如果蛋白质以相同的结合位点以和 off 速率相似的结合位点,则添加竞争蛋白质的顺序不重要。在进行实验之前,需要建立两个控制。首先,每个竞争蛋白应该添加到单独的孔,而不存在His标记的同源蛋白。我们进行了这个控制以确认完全去除His标签并且确认竞争蛋白在没有S6-His存在的情况下不结合板。第二对照是确定在Ni d O 2 + -NTA结合位点变饱和之前给出线性检测范围的His标签蛋白的摩尔量。在该测定中使用的板是具有10-20pmol /孔的报道的结合能力的96孔Ni 2+ 2+ -NTA包被的板。我们测试了5-150 pmol /孔的一系列His标签蛋白量,发现当使用每孔200μl体积时,信号在80pmol饱和。因此,我们使用小于80pmol /孔的S6-His蛋白用于测定,以确保竞争性未标记的S18蛋白的摩尔浓度将超过结合到平板上的His标签蛋白。总是使用新鲜纯化和重折叠的蛋白质(或解冻的等分试样储存在-80℃)和冰冷缓冲液用于所有稀释和洗涤以在整个测定中保持核糖体蛋白质的完整性。

  1. 为了开始该实验,需要纯化的重组蛋白。如果His-tag用于纯化,则竞争性蛋白质(如在S18-1和S18-2的情况下)必须除去其标签,而同源蛋白质/配体即S6蛋白质必须保留His标记。如前所述(Prisic等人,2015),我们对S18-1,S18-2和S6使用六组氨酸标签,并在变性条件下在钴柱上纯化重组蛋白,然后重折叠通过透析。使用工程化的TEV切割位点,我们用TEV蛋白酶从S18-1和S18-2除去组氨酸标签,随后用His标签纯化树脂基质纯化切割的蛋白质。
  2. 确保蛋白质都在相同的缓冲液中;在这种情况下使用MST缓冲液。如果它们不在相同的缓冲液中,则应当重新透析,或者使用凝胶过滤来交换缓冲液。将核糖体蛋白重折叠并保存在MST缓冲液中,其使用微量电泳进行结合研究优化(Prisic等人,2015)。考虑到我们观察到S6和每个S18蛋白在该缓冲液中的强相互作用,我们决定在置换测定中使用相同的条件。组分不干扰Ni 2+ + NTA-His标签结合,并且对ELISA具有可接受的作用,即,一抗和二抗的结合,以及HRP活性。 MST缓冲液用于所有稀释,包括抗体稀释,以及洗涤步骤。我们平行进行该测定,一个用Zn 2+透析的蛋白质,一个用EDTA透析的蛋白质。因此,当使用Zn 2+/sup/- 透析蛋白质或MST与EDTA一起使用时,我们将使用具有Zn 2+ 2+(10μMZnSO 4)的MST 100μMEDTA)时,使用EDTA透析的蛋白质。
  3. 确定蛋白质的浓度。对于核糖体蛋白,我们使用BioRad蛋白浓度测定,改进的Bradford方法。在MST缓冲液中制备BSA标准品,并且当在酶标仪中测量595nm处的吸光度时,从标准曲线确定蛋白质浓度。蛋白质的分子量为S6-His为11.9kDa,S18-1为9.6kDa,S18-2为9.8kDa。使用下式从Bradford测定法给出的蛋白质浓度确定蛋白质的摩尔浓度:

  4. 对于所有待测试的孔,制备在MST中稀释的His-标记蛋白的主混合物。我们使用30 pmol /孔的His标签蛋白和200μl孔体积,因此将蛋白质稀释至150μM的终浓度。等分200微升稀释的His标记蛋白到每个孔(图1a)。建议至少进行两到三次技术重复。
  5. 在室温下,在台式振荡器上以300rpm振荡孵育板30分钟
  6. 从板上弃去溶液,用200μlMST洗涤孔四次。用冰冷的缓冲液洗涤,但在室温下进行。孔用洗涤缓冲液浸泡30-60秒,不摇动,并在每次洗涤之间在纸巾上干燥
  7. 使S6结合配偶体( S18-2蛋白)的主混合物的摩尔浓度比用于His-标记的蛋白质的摩尔浓度至少大1.5倍,因此存在足够的材料以确保完全His标签蛋白的饱和。在我们的情况下,我们使用270μM(54pmol /孔)的S18-2蛋白的摩尔浓度。每孔等分200μl主混合物(图1b)
  8. 在室温下在板振荡器上摇动(300rpm)孵育板30分钟。
  9. 从板上弃去溶液,并按步骤6所述进行清洗。
  10. 以下列方式制备另一种S6结合蛋白(即S18-1)的摩尔浓度范围。以4倍量的步骤7中使用的第一S6结合配偶体(在我们的情况下,其为1.08mM)制备溶液。创建连续稀释,每个步骤减少浓度一半,直到获得0.25x(67.5μM)。
  11. 等分200微升每个稀释每孔。包括一个孔,其中没有竞争蛋白被添加以建立从His标记的同源蛋白的饱和获得的全强度信号,没有位移(图1c)。
  12. 在室温下,在台式振荡器上摇动(300rpm)孵育板30分钟
  13. 从板上弃去溶液,并按步骤6所述进行清洗。
  14. 准备一级抗体(竞争蛋白之一的特异性抗体,在我们的情况下是针对S18-2),在含有3%BSA的MST中以1:1000稀释。每孔200μl抗体溶液等分。
  15. 在室温下,在台式振荡器上摇动(300rpm)孵育板30分钟
  16. 从板上弃去溶液,并按步骤6所述进行清洗。
  17. 使用3%BSA在MST中制备1:5,000稀释的二抗(与HRP缀合)。等分200微升二次抗体溶液到每个孔,包括控件
  18. 在室温下,在台式振荡器上摇动(300rpm)孵育板30分钟
  19. 从板上弃去溶液,并按步骤6所述进行清洗。
  20. 使用多通道移液器将200μl化学发光底物(例如SuperSignal Pico稳定过氧化物和底物的1:1混合物)等分到每个孔中。尝试随着信号随时间快速增加而快速连续地向板的每行(在我们的情况下每行是一个完整的重复)添加底物。使用具有预混基底的槽将允许最小轮移液。最重要的是,板的每一行具有同时使用多通道移液管添加的底物,使得没有竞争蛋白质的孔的标准化和具有竞争每个重复的处理孔具有同时添加的底物。
  21. 轻轻摇动板1分钟,并立即读板化学发光信号(LUM)在读板器(图1d)。
  22. 将每个孔的信号标准化为从其中没有添加竞争蛋白的孔获得的信号,代表完全饱和的信号。从每个处理井获得的信号将表示为在没有竞争的情况下获得的全强度信号的百分比(图1e)。

代表数据


图1.逐步描绘基于置换的ELISA。在置换测定的整个阶段描述通过96孔板的六个孔的虚拟横截面。一个)。通过His-标记将配体(μL)固定在Ni 2+ + -NTA板上。 b)。过量加入第一结合配偶体P1 以饱和由固定的配体提供的结合位点。 C)。将第二结合配偶体P2 以增加的量加入到孔中,所述量从结合到L 的最大理论浓度的0-4X起, 。在发生 P1 发生 P2 的情况下,原来绑定到 的更换为 P2 。被置换的 P1 的量将取决于添加到阱中的P2/P1 的比率,更高的比率导致更少的< em>保持与 L 绑定。 d)。置换测定的结果通过使用特异于P1的一级抗体和偶联辣根过氧化物酶(HRP)的二级抗体的ELISA获得。从发光HRP底物发射的光是浓度依赖性的,并且通过将来自每个孔的信号归一化到其中未引入P2 ^的信号来确定位移。(即 与 的完全饱和)。 e)。位移可以通过在P2/P1比率的测试范围上绘制P1的标准化信号而可视化。该图显示了位移或没有位移发生的两个假设结果。在协议 P1 中描述的情况是S18-2, P2 是S18-1, L 是S6-His。

食谱

  1. 1. 5x HEPES/KCl(1L)
    123 g KCl
    23.83g HEPES钠盐
    900ml H 2 O 2 / 用NaOH调节pH至7.6 使最终体积为1 L
  2. MST缓冲液(20mM HEPES pH7.6,330mM KCl,0.05%Tween 20,14mM 2-巯基乙醇)(1L)
    200ml 5x HEPES/KCl
    2.5ml 20%Tween 20
    1ml 2-巯基乙醇 使最终体积为1 L
    注意: 始终使MST缓冲区更新并使用同一天。
  3. MST与Zn 2 +
    MST缓冲液+10μMZnSO 4
  4. 用EDTA的MST MST缓冲液+100μMEDTA

致谢

这项工作是由夏威夷大学在马诺阿的创业基金支持。

参考文献

  1. Prisic,S.,Hwang,H.,Dow,A.,Barnaby,O.,Pan,T.S.,Lonzanida,J.A.,Chazin,W.J.,Steen,H。和Husson, 锌调节结核分枝杆菌中的原始和替代S18核糖体蛋白之间的转换 。 Mol Microbiol 97(2):263-280。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dow, A. and Prisic, S. (2016). Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate. Bio-protocol 6(5): e1745. DOI: 10.21769/BioProtoc.1745.
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