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May 2019

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Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT
利用Autolab ESPIRIT进行表面等离子体共振分析蛋白间结合特异性   

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Abstract

Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), Surface Plasmon Resonance (SPR) and many more. Among these, SPR stands out as a quick, label-free, reliable, and accurate quantitation technique. We have used SPR to elucidate the linkage between 14-3-3 Protein 3 (EhP3) and the actin cytoskeleton in the protist pathogen Entamoeba histolytica. It allowed us to screen EhP3 binding with several actin-binding/actin regulatory proteins (Coactosin, Actophorin, Twinfilin, Profilin, and Filamin). Our screening results suggested Coactosin as an important interacting partner of EhP3. A complete kinetic analysis indeed confirmed that EhCoactosin binds EhP3 with an affinity constant of 3 μM.

Keywords: SPR (表面等离子体共振), EhP3 (EhP3), 14-3-3 (14-3-3), Entamoeba histolytica (痢疾阿米巴), Cytoskeleton/actin (细胞骨架/肌动蛋白)

Background

Surface Plasmon Resonance (SPR) technique has emerged as one of the most promising screening tools to study macromolecular interactions, since its inception in the early 1990s (Nguyen et al., 2015). It helps determine binding affinities, kinetic parameters, and specificity of these interactions in real-time. This technique is simply based on the optical property of light and principally measures the change in the refractive index upon binding of any molecule to the surface. Surface Plasmon Resonance utilizes the interaction occurring between light and matter. The SPR signal is determined at the surface of the sensor, thus it can be easily correlated to the macromolecules bound on it. This technique captures the real time binding between interacting molecules in a label-free manner, thus making it easy to perform and analyze. Moreover, unlike ITC, this technique is not solution based and uses solid surface for ligand immobilization. SPR uses low quantities of reagents, thus working with small sample size is not an issue.

Principle: Surface Plasmon is a plane-polarized electromagnetic wave that travels on the surface at the interface of the metal coating of the sensor disk (gold disk) and the dielectric medium (sample layer/buffer). These waves are created by continuous fluctuation of charge at the metallic surface. The thin metal surface is placed incident to a laser beam. When the incident light strikes at the interface of the two dielectric media (gold layer and sample layer interface), total internal reflection occurs. Simultaneously, this light generates an evanescent field, with its maximum intensity at the surface of the dielectric material. The resonance occurs when the free electrons of the metal oscillate (plasmon wave) and absorb the plane-polarized light at the specific angle (SPR angle). At the SPR angle, a sharp decrease in the intensity of the reflected light (since some of light is transferred to the Plasmon wave) is observed and this serves as the measurement key. Light passes through a prism and reaches the surface (gold surface) where the molecules are bound. Prism is required to make sure that the wave vector of light in air (kx) is same as the wave vector at the noble metal surface (ksp). The wave vector parallel to the surface is of sole importance in an SPR experiment since the plasmons are confined to the plane of the gold surface. The relationship between kx and incidence angle can be stated  as:

kx = k0 nglass sinθinc

where k0 can be calculated as k0 = 2πλ0-1, nglass is the refractive index of the glass prism and θinc is the angle of incidence.

To make the laser light hit the gold disk surface directly, immersion oil is used between the disk and the hemi-cylinder prism (Figure 1). The refractive index of the prism glass, immersion oil, and the disk are same i.e. 1.5. The sensing surface (gold surface) has the lowest refractive index, thus the position of the SPR angle depends on it. Any change (for example: protein-protein interactions typically have a refractive index increment of about 0.18-0.19 ml/g) (Davis et al., 2000) in the dielectric constant or the refractive index changes the resonance angle thus making the SPR-effect a useful tool for us. As the macromolecules bind the surface, the refractive index changes, changing the SPR angle. This change is directly sensed by the detector and translated into response units. The response units obtained are then used to analyze the binding kinetics. The shift in the SPR angle shares a linear relationship with the amount of sample bound. 120 m° (millidegrees) change represents a change in surface protein mass of ~1 ng/mm2. Since light does not penetrate the sample, any colored, turbid, or opaque sample can be used to study.


Figure 1. Pictorial representation of the Surface Plasmon Resonance principle. The diagram depicts the basic underlying principle of the plasmon wave on the gold surface. The Amine groups and the Thiol groups represent the two surface activation method available. The smiley represents the immobilized ligand protein. The chemical structures are labeled to depict the surface chemistry. ksp represents the surface wave vector and kx is the x component of the light wave vector. Absorbed light represents the light at angle of minimum reflection that is recorded as the absorbance by the detector. Evanescent field is a comparable electric field generated by the plasmons on either side of the surface. SPR angle and the Angle of reflection can be clearly seen as read by the detector.

Instrument: Figure 2A shows the Autolab ESPRIT instrument used for the SPR studies. The machine is simple to handle with easy mechanism of operation. The hemi-cylinder is the prism from where the incident light enters and strikes the gold disk. The cuvette (marked as Channel 1 and 2 in the Figure 2B) above it is where all the solutions dispensed in the 384-well plate are injected over the gold disk for the interaction studies to be measured. The syringe pumps modulate the movement of these solutions in and out of the two needles. The Auto sampler moves the needles to and fro for the same. Channel 1 is experimental whereas Channel 2 serves as the control. Simultaneously, the peristaltic pumps below the rack holding the 384-well plate pump in and out the Running buffer. Figure 2C shows the hemi-cylinder with the gold disk placed on it. The Gold Sensor Disk used here is glass disk coated with a very thin layer of gold. The SPR effect can be observed only in the metals where its electrons can behave like a free electron gas. This means that when these electrons move over the surface, their movement is not dependent on the charge that they leave behind. Thus, the only option of metallic surfaces is restricted to Copper, Aluminium, Silver, and Gold.


Figure 2. Autolab ESPRIT instrument. A. The instrument used in the protocol is labeled here. B. Enlarged view of the cuvette, where the reagents are fed onto the gold sensor disk. C. This figure is used from the Autolab manual. The hemi-cylinder is mounted with the gold disks, as placed inside the machine.

Another instrument, commonly used for SPR studies is Biacore T200, sold by G.E. Healthcare. The difference lies at the mechanism of measurement of the resonance angle. Biacore uses a convergent beam, thereby generating a number of incidence and reflecting angles. The disadvantage of this mechanism is that it decreases the resolution to 10-3 in the refractive index. Autolab ESPRIT on the other hand the incident light can be changed by the use of vibrating mirror system. The critical angle for total internal reflection can be changed using the mirrors and the angular shift is measured for a non-coated gold sensor surface with a resolution of approximately 0.02 millidegrees (m°), corresponding to a refractive index resolution of approximately 1 x 10-5. For a coated gold sensor surface, the angular shift is measured with a resolution of approximately 0.1 millidegrees (m°); corresponds to 2 x 10-6 refractive index resolution.

The Chemical steps
Baseline: The first step is to measure the response units for the coupling buffer on the gold sensor disk and is done by simple passing the coupling buffer over the gold surface. It helps in normalization of the further measurements to subtract the noise from the buffer.

Activation: In this step (Figure 3) the gold surface is chemically activated so that protein can be conjugated to it. It can be done by several ways, the most commonly used for example, are: Amine coupling: With the use of EDC (Dimethylaminopropyl-N′-Ethylcarbodiimide N-3-hydrochloride) and NHS (N-Hydroxy Succinimide), the surface can be activated followed by attachment of the protein through its NH2 group. The final concentrations used are 400 mM and 100 mM for EDC and NHS respectively. Thiol coupling: If the ligand protein has a thiol group, then the surface can be made active by using PDEA (2-(2-pyr-idinyldithio) ethaneamine hydrochloride)).

Coupling: At this step, the protein is injected and allowed to bind to the activated surface. This step is termed as immobilization of the ligand protein. As the ligand protein binds, the SPR angle changes linearly with increase in concentration and is measured in its response units.

Blocking: A blocking compound, essentially Ethanolamine here, is added to coat any remaining activated site on the sensor to prevent non-specific binding during the SPR reaction. The blocked sites will be as shown in Figure 1.

Regeneration: This is primarily washing off of any unbound chemical and to set the new baseline value after protein immobilization.


Figure 3. Gold surface activation reactions for the sensor disks. A. The steps for the amine coupling of the ligand protein to the gold surface. B. The steps for the thiol coupling of the ligand protein, using respective chemical reagents. The smiley represents the ligand protein to be immobilized.

Once these steps are successfully performed, the analyte is then passed over the ligand protein to perform the SPR assay. The following Figure 4. represents a typical layout of the reaction and its steps. Here, the running buffer (Association Buffer) is used to set the baseline value since the analyte is prepared in this buffer. As the analyte is injected, the response units increase upon its binding to the ligand protein. Once it reaches a steady state, the dissociation kinetics is measured by just flowing the running buffer over the sensor chip, allowing the analyte to dissociate from the bound ligand. Finally, the regeneration buffer is used to remove the bound analyte to free the ligand protein for the next set of binding reactions.


Figure 4. Steps in a typical SPR assay. Running buffer serves as the baseline for the entire reaction. The Response Units (RU) measured for the running buffer is used to normalize all the calculations. If the injected analyte binds to the ligand protein, then the increase in RU is recorded, while the dissociation of the same analyte leads to the decreasing RU. Regeneration buffer removes any bound analyte (if any) and prepares the ligand for the next round of association reaction.

Relative to other established methods for monitoring protein-protein interactions; SPR-based techniques allow easy experimental designs, rapid testing of hypotheses with high sensitivity, and accurate quantification. In a recent study published by us, SPR was used to determine the interacting partners for EhP3, in order to decipher its connection with the actin cytoskeletal system in Entamoeba histolytica. We conclusively found that EhCoactosin binds EhP3 with an affinity constant of 3 μM (Kumar et al., 2014; Agarwal et al., 2019). This protocol is a one-step handout for anyone who wishes to perform SPR using the Autolab ESPRIT machine. We have explained all the steps in an elaborate manner to help the user to carryout a hassle free experiment. The mechanisms of this instrument are different from Biacore T200 and this protocol can be easily followed by, even a first time user.

Materials and Reagents

  1. Bare gold sensor disk (Metrohm Autolab, catalog number: 1-04-04-000)
  2. 384-well Clear Flat Bottom Polystyrene Not Treated Microplate, with Lid, Sterile (Corning, catalog number: 3680)
  3. 6-well Clear Not Treated Multiple Well Plates (Corning Costar, catalog number: 3736)
  4. Immersion oil
  5. Demineralized (demi) water (Sigma-Aldrich, catalog number: 38796)
  6. Mercaptoundecanoic acid (11-MUA) (Sigma-Aldrich, catalog number: 450561, MW 218.36 g/Mol)
  7. N-Hydroxy Succinimide (NHS) (Sigma-Aldrich, catalog number: 130672, MW 115.09 g/Mol)
  8. Dimethylaminopropyl-N′Ethylcarbodiimide N-3-hydrochloride (EDC) (Sigma-Aldrich, catalog number: 03450, MW 191.70 g/Mol)
  9. Ethanolamine (Sigma-Aldrich, catalog number: E6133, MW 97.54 g/Mol)
  10. Sodium acetate trihydrate (Sigma-Aldrich, catalog number: S8625, MW 136.08 g/Mol)
  11. Acetic acid (Sigma-Aldrich, catalog number: A6283)
  12. HEPES sodium salt (Sigma-Aldrich, catalog number: H7006, MW 260.29 g/Mol)
  13. Ethanol, ≥ 99.9% (Merck, catalog number: 100983)
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653, MW 58.44 g/Mol)
  15. Sodium hydroxide (NaOH) (Merck, catalog number: 1.06498, MW 40.00 g/Mol)
  16. Hydrochloric acid (HCl), 37% (Merck, catalog number: 100317)
  17. Tween 20 (Sigma-Aldrich, catalog number: P1379)
  18. Nitrogen gas (Sigma-Aldrich, catalog number: 295574)
  19. 11-MUA solution (see Recipes)
  20. 400 mM EDC (see Recipes)
  21. 100 mM NHS (see Recipes)
  22. 1 M Ethanolamine (see Recipes)
  23. 1 M Acetate buffer (see Recipes)
  24. 1 M HEPES buffer (see Recipes)
  25. 5 M NaCl (see Recipes)
  26. 1 M NaOH (see Recipes)
  27. 10% Tween 20 (see Recipes)
  28. Buffer 1: Coupling buffer (see Recipes)
  29. Buffer 2: Association buffer (see Recipes)
  30. Buffer 3: Regeneration buffer (see Recipes)
  31. Protein solutions (see Recipes)

Equipment

  1. Tweezers
  2. Pipettes
  3. Autolab SPR ESPRIT (Metrohm, model: Autolab ESPRIT)
  4. Tabletop high-speed centrifuge (Eppendorf, model: 5430R)
  5. Filtration unit (Milipore, model: XX1514700, and simple Syringe filters)

Software

  1. Autolab Data Acquisition program
  2. Autolab Kinetic Evaluation program

Procedure

  1. Preparation of the gold disk sensor (Figure 5)
    1. Place the bare gold disk carefully in a 6-well plate using a pair of tweezers, keeping the gold surface facing upwards.
    2. Add the solution of 11-MUA (Recipe 1) and cover the plate carefully.
    3. Incubate the disk overnight in this solution to obtain a uniform layer of thiols over the gold surface. The plate is covered with foil and incubated at room temperature.
    4. Next day, wash three times with ethanol to remove excess thiol groups. For this, pipette out the 11-MUA solution and pipette in ethanol. The volume of the ethanol should be just enough to completely immerse the disk. Gently shake the plate to rinse the disk.
    5. To remove ethanol, wash thrice with demineralized water, while gently shaking the plate for rinsing the gold disk.
    6. After thorough washing, blow-dry the disk by flushing the well with nitrogen gas.
    7. These disks can now be stored up to 2 months in their container at room temperature.


      Figure 5. Preparation of the gold sensor disk. The diagram explains all the steps for the Steps A1 to A7 as mentioned in the text.

  2. Setting up the sensor disk (Figure 6)
    1. Put a small drop of the immersion oil on the outer edge of the hemi-cylinder.
    2. Place the 11-MUA coated gold disk on the other end using a pair of tweezers. Now, simply push the disk carefully by sliding till it is correctly set on the hemi-cylinder.
    3. Make sure the gold surface is facing upwards.
    4. No air bubble should be present between the disk and the immersion oil on the hemi-cylinder.


      Figure 6. Setting up the gold disk on the hemi-cylinder. B1. The hemi-cylinder is prepared by adding a drop of immersion oil on it. B2. The coated gold disk is placed on the oil drop and slid through using a pair of tweezers.

  3. Immobilization of the protein (Figure 7)
    1. Pass Buffer 1 (Coupling Buffer) over the modified gold disk. This hydrates the gold surface and stabilizes the thiol layer (Recipe 10).
    2. In the 384-well microplate, dispense out all the solutions required for the immobilization procedure into the wells of the column 1 as marked in Figure 7.
    3. Add 80 μl EDC (400 mM) in wells 1A and 1B, followed by 80 μl of NHS (100 mM) in both the wells 1C and 1D.
    4. Leave 1E and 1F blank for mixing of EDC and NHS. This mixture will activate the gold sensor disk for ligand protein immobilization.
    5. Add 80 μl of the ligand protein (example: 5 mg/ml of EhP3) in both 1G and 1H wells.
    6. Finally, add 80 μl of Ethanolamine (1 M) in 1I and 1J, to be used for blocking the remaining unbound area on the sensor.
    7. Place the 384-well plate onto the rack as shown in Figure 1.
    8. Use the following steps for setting the data acquisition times in the Data Acquisition Software:
      Steps Time
      Baseline (Coupling buffer)
      120 s
      Activation (EDC/NHS)
      300 s
      Coupling (Ligand protein)
      900 s
      Blocking (Ethanolamine)
      600 s
      Regeneration (coupling buffer)
      120 s


      Figure 7. 384-well plate used in this protocol. Each well used in the plate is marked here.

  4. Interaction studies
    1. In column 2 of the 384-well microplate, add different dilutions of the Analyte, i.e., protein (or different proteins) to be used for determining their association kinetics.
    2. In well 2A, add 80 μl of Analyte 1 (example: Filamin) [or one dilution (1 μM) of a single protein (example: EhCoactosin)] followed by 80 μl of Buffer 2 (Association Buffer) (Recipe 11) in well 2B as reference.
    3. Similarly, add 80 μl of all the Analytes (examples: Actophorin, Profilin A, Profilin B, Twinfilin, EhCoactosin) or (examples: 2 μM, 4 μM, 8 μM, 10 μM of EhCoactosin) into alternative wells, followed by Buffer 2 (Association buffer) in each of the consecutive wells.
    4. Use Buffer 2 as the running buffer for the assay.
    5. Use the following incubation times for interaction studies:
      Steps
      Time
      Baseline
      120 s
      Association
      300 s
      Dissociation
      300 s
      Regeneration
      120 s
    6. The SPR assays also use controls like any other protein-protein interaction study. Bovine Serum Albumin generally serves as the negative control, i.e., unless the protein in focus binds to it specifically. BSA normally does not bind majority of the proteins. Similarly, if any known interacting protein is known, then it can be used as the positive control. Further, the buffers used in the study should be compatible to all the proteins. This can be checked by diluting a small amount of the proteins in all the buffers to be used, and check them for any precipitation.

Data analysis

Use the AutoLab Kinetic Evaluation software to obtain the ‘Differential curves’ (Figure 8). Use these to determine the Kon and Koff, along with with the affinity constant KD. The strength of interaction between two molecules is said to be stronger if the display a smaller KD value. The higher value of KD signifies weaker attraction between the interacting molecules. Kon represents the association constant with units in min-1 multiplied by concentration-1. Koff is the dissociation constant with units in min-1. Affinity constant is determined by the ratio of Koff/Kon in Molar units.
The Video 1 can be seen for data analysis steps.

  1. Open the Kinetic Evaluation Software.
  2. Follow the steps shown in the Video 1.

    Video 1. Steps to use the Kinetic Evaluation Software

  3. Select the folder where the files from the Data Acquisition software have been saved.
  4. In the folder, select all the series of injections recorded for the protein interaction study.
  5. On the left hand top corner, under the heading ‘Create Files’, click New Overlay and select all the injections to be used for the kinetic analysis.
  6. After this click on Signal Processing tab on the left hand bottom corner. Click on zoom and select the region of the sensogram before the association curve. Select Synchronize under the drop down tab of ‘Actions’. Next, click on ‘Normalize’ under the same tab and apply.
  7. To remove the regeneration curves, click on ‘Delete Selection’ and select the region after the dissociation curve.
  8. Again click on Normalize.
  9. Close the tab, and open New project under ‘Create Files’ and save the data with appropriate name.
  10. Next, open the Analysis wizard, select Equilibrium, click next. After this feed in the respective concentration.
  11. Finally, click next and the calculations for the kinetic parameters can be performed successfully.


    Figure 8. SPR analyses of cytoskeletal proteins to immobilized EhP3. A. Surface Plasmon Resonance was used to determine the interaction between EhP3 and cytoskeletal proteins like Filamin, Actophorin, Profilin A and B, Twinfilin, and EhCoactosin. EhP3 was immobilized covalently through amide coupling onto the gold sensor. Shown here are the sensorgrams of various proteins injected at 200 nM concentrations. B. Based on the sensograms observed in (A), we have depicted the interaction in this cartoon model, in which, only EhCoactosin showed binding to EhP3. The other proteins could not bind to the immobilized protein EhP3. C. Shown here are sensorgrams of EhCoactosin injections of increasing concentration. The equilibrium binding curves were used to derive the affinity constant KD. The concentrations of recombinant His6-Ehcoactosin are indicated alongside the binding curves

Notes

  1. The ideal buffer for proteins to be used for SPR studies would be HEPES, but if the protein stability is an issue, then keep the concentration of any other buffer to a maximum of 50 mM.
  2. EDC and NHS should be preferably freshly prepared as they are not very stable in solution. If prepared in larger quantity, one should store them as 300 μl aliquots in -20 °C for as long as thawed next time. However, it is not recommended to store them after thawing once.
  3. Preparation of the ligand in acetate buffer should always be done fresh. Moreover, the same acetate buffer stock should be maintained during the entire immobilization procedure.
  4. The pH of the coupling buffer can vary in case of different proteins. A general equation can be used to determine the pH of the coupling buffer: pH (buffer) = pI (ligand protein) - 0.5. pI is the isoelectric pH of the protein at which all its negative charges are equal to all its positive charges, making it electrically neutral. It can be theoretically calculated for any protein with known amino acid composition.
  5. There exists a linear relationship between the shift in SPR angle with the amount of the bound material. 120 m° change represents a change in surface protein mass of ~1 ng/mm2.
  6. Most kinds of organic solvents are not recommended for this machine, kindly refrain from using those in any of the sample preparations.
  7. Acetate buffer, with its lowest refractive index, serves as the best buffer for the immobilization process, as it gives the smallest SPR angle. Ethanolamine solution records the largest SPR angle.
  8. For any new protein-protein interaction, normally one can decide the concentration by referring to the available data on those proteins, if any. Otherwise, one can begin by taking a wide range of concentrations, example 50 nM-500 nM. If this range gives no information, one can increase the range up to 2 μM. Based on these reaction curves, ideal concentrations can be chosen for the particular set of proteins.
  9. Maintenance of the instrument:
    1. Hemi-cylinder should be regularly cleaned due to inevitable spillage of the immersion oil. As shown in Figure 2, M3X3 screw should be carefully removed and the hemi-cylinder should be cleaned with either ethanol or in an ultrasonic water bath. To dry the apparatus, ONLY lens tissue must be used. After the cleaning and drying, the hemi-cylinder must be placed and screwed back on the slider.
    2. In order to avoid any evaporation of solution from the cuvette, cover it with Para film.
    3. Always clean the instrument with distilled water. Use of any kind of buffer will lead to salt deposition and thus rendering the coating useless.
    4. The syringe pump seals should be changed on a yearly basis for optimum performance of the instrument.

Recipes

  1. 11-MUA solution
    11 mg mercaptoundecanoic acid
    50 ml ethanol
    Filter sterilize the solution for optimum results
  2. 400 mM EDC (2 ml)
    153.4 mg EDC
    2 ml demi water
  3. 100 mM NHS
    23 mg NHS
    2 ml demi water
  4. 1 M Ethanolamine
    0.97 g salt
    9 ml demi water
    Use HCl to adjust its pH to 8.5
    Finally, make up the volume to 10 ml
  5. 1 M Acetate buffer pH 4.5
    1.36 g salt
    9 ml demi water
    Use acetic acid to adjust the pH to 4.5
    Finally, make up the volume to 10 ml (Abdul Rehman et al., 2013)
  6. 1 M HEPES buffer pH 7.5
    26.03 g salt
    80 ml demi water
    Use HCl to adjust the pH to 7.5
    Finally, make up the volume to 100 ml
  7. 5 M NaCl
    29.22 g salt
    100 ml demi water
  8. 1 M NaOH
    0.4 g of NaOH
    10 ml demi water
  9. 10% Tween 20
    Prepared with demi water
  10. Buffer 1 (Coupling buffer)
    Dilute 0.5 ml of Acetate buffer stock to 50 ml solution using demi water
  11. Buffer 2 (Association buffer) (1 L)
    10 ml 1 M HEPES buffer pH 7.5
    30 ml NaCl from its stock solution
    0.5 ml 10% Tween 20
    Make up the final volume to 1 L with demi water
    Filter and use
  12. Buffer 3 (Regeneration buffer)
    Take 2.5 ml from NaOH stock solution
    Make up the volum
  13. Protein solutions
    1. Convert mg/ml protein concentration to molarity. Use the following formula:
      μM of protein = (μg/μl of protein)/(MW in Da)
    2. Next, dilute the protein stock in Buffer 2 to prepare 100 μM of working stock.
    3. Use the following calculations to prepare the dilution in Buffer 2 (Association buffer):
      Sample name
      Volume of protein working stock
      Volume of Buffer 2
      Dilution 1 (1 μM)
      1 μl
      99 μl
      Dilution 2 (2 μM)
      2 μl
      98 μl
      Dilution 3 (4 μM)
      4 μl
      96 μl
      Dilution 4 (8 μM)
      8 μl
      92 μl
      Dilution 5 (10 μM)
      10 μl
      90 μl
    4. When using different proteins against one protein, use the same dilution (made in Buffer 2) for all the proteins. Make 1 μM dilution of all the proteins to be used as working stock. Take 20 μl of this stock and add 80 μl of Buffer 2 to prepare 200 nM solutions.
      Protein name
      Concentration
      Analyte 1 (example: EhCoactosin)
      200 nM
      Analyte 2 (example: Profilin A)
      200 nM
      Analyte 3 (example: Profilin B)
      200 nM
      Analyte 4 (example: Filamin)
      200 nM
      Analyte 5 (example: Twinfilin)
      200 nM
      Analyte 6 (example: Actophorin)
      200 nM

Acknowledgments

We acknowledge Advanced Instrument Research Facility, JNU and Manu Vashistha for SPR studies. We would also like to thank Prof. Alok Bhattacharya (Ashoka University, New Delhi, India) and Prof. Samudrala Gourinath (Jawaharlal Nehru University, New Delhi, India) for providing us the necessary help during the study. SA thanks the Department of Science and Technology for DST Inspire Faculty Award and Grant. PPR thanks the Council of Scientific and Industrial Research (CSIR) for SRF Fellowship.

Competing interests

The authors have declared that no competing interests exist.

References

  1. Abdul Rehman, S. A., Verma, V., Mazumder, M., Dhar, S. K. and Gourinath, S. (2013). Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori. J Bacteriol 195(12): 2826-2838.
  2. Agarwal, S., Anand, G., Sharma, S., Parimita Rath, P., Gourinath, S. and Bhattacharya, A. (2019). EhP3, a homolog of 14-3-3 family of protein participates in actin reorganization and phagocytosis in Entamoeba histolytica. PLoS Pathog 15(5): e1007789.
  3. Davis, T. M. and Wilson, W. D. (2000). Determination of the Refractive Index Increments of Small Molecules for Correction of Surface Plasmon Resonance Data. Anal Biochem 284: 348-353.
  4. Kumar, N., Somlata, Mazumder, M., Dutta, P., Maiti, S. and Gourinath, S. (2014). EhCoactosin stabilizes actin filaments in the protist parasite Entamoeba histolytica. PLoS Pathog 10(9): e1004362.
  5. Nguyen, H. H., Park, J., Kang, S. and Kim, M. (2015). Surface plasmon resonance: a versatile technique for biosensor applications. Sensors (Basel) 15(5): 10481-10510.

简介

[摘要] 已知直接的蛋白质-蛋白质相互作用可调节广泛的细胞活性。要了解这些接触,可以采用各种实验方法,例如动态光散射(DLS),荧光共振能量转移(FRET),等温滴定热量法(ITC),化学交联,共免疫沉淀(Co-IP),表面等离子体共振(SPR) ) 还有很多。其中,SPR是一种快速,无标签,可靠且准确的定量技术。我们已经使用SPR阐明了原生病原体Entamoeba中14-3-3蛋白3(EhP3)和肌动蛋白细胞骨架之间的联系 组织溶菌。它使我们能够筛选与几种肌动蛋白结合/肌动蛋白调节蛋白(辅肌动蛋白,Actophorin ,Twinfilin ,Profilin和Filamin)结合的EhP3 。我们的筛选结果表明辅肌动蛋白是EhP3 的重要相互作用伴侣。一个完整的动力学分析确实证实了EhCoactosin 结合EhP3 3的亲和常数μM 。

[背景 ] 表面等离子体共振(SPR)技术已成为最有前途的筛选工具来研究大分子相互作用之一,自上世纪90年代初以来(阮等人,2015年)。它有助于实时确定结合亲和力,动力学参数和这些相互作用的特异性。该技术仅基于光的光学特性,并且主要测量在任何分子与表面结合后的折射率变化。表面等离子体共振利用了光与物质之间发生的相互作用。SPR信号是在传感器表面确定的,因此可以很容易地与绑定在其上的大分子相关。该技术以无标记的方式捕获相互作用分子之间的实时结合,从而使其易于执行和分析。而且,与ITC不同,该技术不是基于溶液的,而是使用固态表面进行配体固定。SPR使用少量试剂,因此处理小样本量不是问题。

原理:表面等离激元是一种平面极化电磁波,在传感器盘(金盘)的金属涂层和介电介质(样品层/缓冲液)的界面上传播。这些波是由金属表面上电荷的连续波动产生的。放置薄金属表面,使其入射激光束。当入射光入射到两个介电介质的界面(金层和样品层的界面)时,会发生全内反射。同时,该光产生了一个van 逝场,其最大强度出现在电介质材料的表面。当金属的自由电子振荡(等离子体激元波)并以特定角度(SPR角)吸收平面偏振光时,就会发生共振。在SPR角处,可以观察到反射光强度的急剧下降(因为一些光被传输到等离激元波),这是测量的关键。光穿过棱镜并到达结合分子的表面(金表面)。需要棱镜以确保空气中光的波矢(k x )与贵金属表面的波矢(k sp )相同。平行于表面的波矢量在SPR实验中非常重要,因为等离激元被限制在金表面的平面上。k x 和入射角之间的关系可以表示为:



ķ X = K 0 Ñ 玻璃SINθ INC



其中,k 0 可以为k来计算0 =2πλ 0 -1 ,Ñ 玻璃是玻璃棱镜的折射率和θ INC 是入射角。

为了使激光直接照射到金盘表面,请在盘和半圆柱棱镜之间使用浸油(图1)。棱镜玻璃,浸油和圆盘的折射率相同,即1.5。感测表面(金表面)的折射率最低,因此SPR角的位置取决于它。的任何变化(例如:蛋白-蛋白相互作用通常具有的折射率增量约0.18 0 19毫升/克)(戴维斯等人,2000)在介电常数或折射率改变从而使共振角的SPR效应对我们来说是一个有用的工具。随着大分子与表面的结合,折射率发生变化,从而改变了SPR角。该变化直接由检测器感测并转化为响应单位。然后将获得的反应单位用于分析结合动力学。SPR角的偏移与绑定的样本量具有线性关系。120 m°(毫度)变化表示〜1 ng / mm 2的表面蛋白质量变化。由于光不会穿透样品,因此可以使用任何有色,混浊或不透明的样品进行研究。







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图1.表面等离子共振原理的图示。该图描绘了金表面上的等离激元波的基本基本原理。胺基和硫醇基代表两种可用的表面活化方法。笑脸代表固定的配体蛋白。标记化学结构以描述表面化学。k sp 表示表面波矢量,k x 是光波矢量的x分量。吸收的光表示最小反射角的光,该光记录为检测器的吸收率。van逝场是由表面两侧的等离激元产生的可比较电场。检测器读取时可以清楚地看到SPR角和反射角。



仪器:图2A显示了用于SPR研究的Autolab ESPRIT仪器。机器操作简单,操作机制简单。半圆柱体是棱镜,入射光从该棱镜进入并撞击金盘。上方的比色皿(在图2B中标记为C 通道1和2)是将分配在384孔板中的所有溶液注入到金圆盘上方以进行相互作用研究的方法。注射泵调节这些溶液进出两个针头的运动。自动进样器会将针头来回移动。通道1是实验性的,而通道2作为对照。同时,机架下方的蠕动泵将384孔板泵保持在进出缓冲液中。图2C显示了带有金盘的半圆柱体。这里使用的金感测器盘是玻璃盘,表面涂有非常薄的金层。只能在金属中电子的行为类似于自由电子气的金属中观察到SPR效应。这意味着,当这些电子在表面上移动时,它们的移动不取决于它们留下的电荷。因此,金属表面的唯一选择仅限于铜,铝,银和金。



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图2. Autolab ESPRIT仪器。答:协议中使用的仪器在此处标记。B.试管的放大视图,其中试剂被送入金传感器盘。C.此图从Autolab 手册中使用。半圆筒安装在机器内部的金盘上。



通常用于SPR研究的另一种仪器是GE Healthcare销售的Biacore T200。区别在于共振角的测量机制。Biacore 使用会聚光束,从而产生许多入射角和反射角。该机制的缺点在于其将分辨率降低到折射率的10 -3 。另一方面,Autolab ESPRIT可以通过使用振动镜系统来改变入射光。可以使用反射镜更改全内反射的临界角,并针对分辨率约为0.02毫米(m°)的未镀膜金传感器表面测量角移,对应于约1 x 10 的折射率分辨率-5 。对于镀金传感器表面,角位移的测量分辨率约为0.1毫米(m°)。对应于2 x 10 -6 折射率分辨率。









化学步骤

基线:第一步是测量金传感器盘上耦合缓冲的响应单位,方法是简单地将耦合缓冲移过金表面。它有助于进一步测量的标准化,以从缓冲区中减去噪声。

活化:在此步骤(图3)中,金表面被化学活化,因此蛋白质可以与之结合。它可以通过几种方法中,最常用的例如来完成,包括:胺偶合:随着使用EDC(二甲氨基丙基-N ' - 乙基碳二亚胺N-3-盐酸盐)和NHS(N-羟基琥珀酰亚胺),该表面可以激活后,蛋白质通过其NH 2 基团附着。EDC和NHS 的最终浓度分别为400 mM和100 mM。硫醇偶联:如果所述配体蛋白具有硫醇基团,那么表面可以由通过使用PDEA(2-(2- pyr-活性idinyldit HIO)乙胺盐酸盐)) 。

偶联:在此步骤中,将蛋白质注射并使其结合到活化的表面。该步骤称为配体蛋白的固定。随着配体蛋白的结合,SPR角随浓度的增加而线性变化,并以其响应单位进行测量。

封闭:添加一种封闭化合物,在这里主要是乙醇胺,以覆盖传感器上任何剩余的活化位点,以防止SPR反应期间发生非特异性结合。被阻止的站点将如图1所示。

回复:这主要是洗的掉任何未结合化学和设置蛋白质固定后的新基准值。



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图3.传感器磁盘的金表面活化反应。A.将配体蛋白与金表面进行胺偶联的步骤。B.使用相应的化学试剂进行配体蛋白的硫醇偶联的步骤。笑脸代表要固定的配体蛋白。



一旦成功执行了这些步骤,分析物便会通过配体蛋白,以执行SPR分析。下面˚F igure 4.代表反应和其步骤的典型布局。在此,由于在该缓冲液中准备了分析物,因此使用运行缓冲液(Association Buffer)来设置基线值。当注射分析物时,响应单元在其与配体蛋白结合时增加。一旦达到稳定状态,就可以通过使运行缓冲液流过传感器芯片来测量解离动力学,从而使分析物从结合的配体上解离。最后,再生缓冲液用于去除结合的分析物,以释放配体蛋白,用于下一组结合反应。



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图4.典型SPR测定中的步骤。运行缓冲液用作整个反应的基线。为运行缓冲区测量的响应单位(RU)用于归一化所有计算。如果注射的分析物与配体蛋白结合,则记录到RU的增加,而相同分析物的解离导致RU的减少。再生缓冲液可去除任何结合的分析物(如果有),并为下一轮缔合反应做准备。



相对于其他建立的监测蛋白质间相互作用的方法;基于SPR的技术允许简单的实验设计,具有高灵敏度的假设的快速测试以及准确的定量。在美国公布的一项最新研究,SPR被用来确定EhP3相互作用的合作伙伴,以破译它与肌动蛋白细胞骨架系统连接阿米巴。我们得出结论发现EhCoactosin 结合EhP3与3的亲和常数μM (库马尔等人,2014;阿加瓦尔等人,2019)。对于希望使用Autolab ESPRIT机器执行SPR的任何人,此协议都是一步式讲义。我们已经解释所有的步骤以复杂的方式来帮助用户结转库存一个无忧无虑的实验。该仪器的机制不同于Biacore T200,即使是初次使用的用户也可以轻松遵循此协议。

关键字:表面等离子体共振, EhP3, 14-3-3, 痢疾阿米巴, 细胞骨架/肌动蛋白

材料和试剂


 


裸金传感器磁盘(Metrohm Autolab ,目录号:1-04-04-000)
384孔透明平底聚苯乙烯未经处理的微孔板,带盖,无菌(Corning,目录号:3680)
6孔未处理的透明多孔板(Corning Costar,目录号:3736)
浸油
脱矿质水(Sigma-Aldrich,目录号:38796)
巯基十一烷酸(11-MUA)(Sigma-Aldrich,目录号:450561,MW 218.36 g / Mol)
N-羟基琥珀酰亚胺(NHS)(Sigma-Aldrich,目录号:130672,MW 115.09 g / Mol)
二甲氨基丙基-N ' 碳二亚胺N-3-盐酸盐(EDC)(Sigma-Aldrich,目录号:03450,MW 191.70 g / Mol)
乙醇胺(Sigma-Aldrich,目录号:E6133,MW 97.54 g / Mol)
三水合乙酸钠(Sigma-Aldrich,目录号:S8625,MW 136.08 g / Mol)
乙酸(Sigma-Aldrich,目录号:A6283)
HEPES钠盐(Sigma-Aldrich,目录号:H7006,MW 260.29 g / Mol)
乙醇 ≥ 99.9%(Merck公司,目录号:100983)
氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653,MW 58.44 g / Mol)
氢氧化钠(NaOH)(Merck,目录号:1.06498,MW 40.00 g / Mol)
盐酸(HCl),37%(Merck,目录号:100317)
吐温20(Sigma-Aldrich,目录号:P1379)
氮气(Sigma-Aldrich,目录号:295574)
11-MUA解决方案(请参阅食谱)
400 mM EDC(请参阅食谱)
100 mM NHS(请参阅食谱)
1 M乙醇胺(请参阅食谱)
1 M醋酸盐缓冲液(请参阅食谱)
1 M HEPES缓冲区(请参阅食谱)
5 M NaCl(请参阅食谱)
1 M NaOH(请参阅食谱)
1 0%补间20 (请参阅食谱)
缓冲区1:耦合缓冲区(请参见配方)
缓冲区2:关联缓冲区(请参阅食谱)
缓冲区3:再生缓冲区(s 配方)
P rotein解决方案(见食谱)
 


设备


 


镊子
移液器
的Autolab SPR ESP RIT(万通,米Odel等:AUTOLAB ESPRIT)
桌面高速离心机仪(Eppendorf,米Odel等:5430R)
过滤单元(Milipore ,米Odel等:XX1514700,和简单的注射器过滤器)
 


软件


 


Autolab 数据采集程序
Autolab 动力学评估程序
 


程序


 


金盘传感器的准备(图5)
用镊子将裸金盘小心地放在6孔板中,并使金表面朝上。
加入11-MUA的溶液(配方1)并小心地盖好板。
在该溶液中将圆盘孵育过夜,以在金表面上获得均匀的硫醇层。将该板用箔覆盖并在室温下孵育。
第二天,用乙醇洗涤三遍,以去除多余的硫醇基团。为此,用移液管吸出11-MUA溶液并用乙醇吸管。乙醇的体积应足以完全浸没磁盘。轻轻摇动板以冲洗磁盘。
要除去乙醇,请用软化水洗涤三次,同时轻轻摇动板以冲洗金片。
彻底清洗后,通过用氮气冲洗孔来吹干圆盘。
现在,这些磁盘在室温下最多可以在其容器中存储2个月。
 


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图5.准备金传感器盘。该图说明了文本中提到的步骤A1至A7的所有步骤。


 


设置传感器盘(图6)
在半圆筒的外边缘上放一小滴浸油。
用镊子将11-MUA涂层金盘放在另一端。现在,只需轻轻滑动磁盘,直到将其正确安装到半圆柱体上即可。
确保金表面朝上。
圆盘和半圆柱体上的浸油之间应没有气泡。
 


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图6.在半圆柱体上设置金盘。B1。通过在其上添加一滴浸油来制备半圆筒。B2。将镀金的圆盘放在油滴上,然后用镊子将其滑过。


 


固定蛋白质(图7)
将缓冲区1(耦合缓冲区)传递到修改后的金盘上。这会水化金表面并稳定硫醇层(配方10)。
在384孔微孔板中,将固定过程所需的所有溶液分配到第1列的孔中,如图7所示。
添加80 微升EDC(400 中的孔1A和1B毫摩尔),随后在80 微升NHS的(100 毫摩尔)的两个孔1C和1D。
将1E和1F留空以混合EDC和NHS。该混合物将激活金传感器盘,以固定配体蛋白。
在1G和1H孔中添加80μl 配体蛋白(例如:5 mg / ml EhP3)。
最后,在1I和1J中添加80μl 乙醇胺(1 M),用于阻塞传感器上的剩余未结合区域。
如图1所示,将384孔板放在架子上。
使用以下步骤在数据采集软件中设置数据采集时间:
步骤时间             


基线(耦合缓冲区)120 s             


激活(EDC / NHS)300秒             


偶联(配体蛋白)900 s             


阻断(乙醇胺)600 s             


再生(耦合缓冲)120 s             


 


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图7。此协议中使用的384孔板。板中使用的每个孔均在此处标记。


 


互动研究
在384孔微孔板的第2列中,添加分析物的不同稀释液,即,将被用于确定其关联动力学蛋白(或不同的蛋白)。
在井2A中,添加80 微升分析物1(的Ë xample:细丝蛋白)[ 或一个稀释(1 μ 中号的单一蛋白质的)(Ë xample:EhCoactosin )] ,随后在80 微升缓冲液2(协会缓冲液)的(配方11 )作为参考。
类似地,添加80 微升的所有分析物的(Ë xamples:Actophorin ,profilin的A,profilin的B,Twinfilin ,EhCoactosin )或(Ë xamples:2 μ 中号,4 μ 中号,8 μ 中号,10 μ 中号的EhCoactosin )转换成替代孔,然后在每个连续的孔中添加缓冲液2(关联缓冲液)。
使用缓冲液2作为分析的运行缓冲液。
使用以下孵育时间进行相互作用研究:
步骤时间             


基线120秒             


关联300 s             


离解300 s             


再生120 s             


SPR分析还像其他任何蛋白质-蛋白质相互作用研究一样使用对照。牛血清白蛋白通常用作阴性对照,即,除非在焦点结合于它的蛋白特异性。BSA通常不结合大多数蛋白质。同样,如果已知任何已知的相互作用蛋白,则可以将其用作阳性对照。此外,研究中使用的缓冲液应与所有蛋白质相容。这可以通过在所有要使用的缓冲液中稀释少量蛋白质来检查,并检查是否有沉淀。




数据分析


 


使用AutoLab Kinetic Evaluation软件获得“差异曲线”(图8 )。用这些来确定K个上和ķ 关闭,随着与亲和常数KD。如果显示的KD值较小,则两个分子之间的相互作用强度据说会更高。KD值越高,表示相互作用的分子之间的吸引力越弱。K on 表示缔合常数,单位为min -1 乘以浓度-1 。K off 是解离常数,单位为min -1 。亲和常数由摩尔单位的K off / K on 的比率确定。


的V 记意1 可以看出对数据进行分析的步骤。


打开动力学评估软件。
遵循视频1中显示的步骤。
 






视频1.使用动力学评估软件的步骤


 


选择从Data Acquisition软件中保存文件的文件夹。
在文件夹中,选择为蛋白质相互作用研究记录的所有注射系列。
在左侧上角,标题下的“创建文件”,单击新的覆盖,并选择要用于动力学分析的所有注射。
在此之后点击信号处理选项卡上的左侧底角。单击缩放并选择关联曲线之前的感觉图区域。在“操作” 的下拉标签下选择同步。接下来,点击同一标签下的“规范化”并应用。
要删除再生曲线,请单击“删除选择”,然后选择解离曲线后的区域。
再次单击规范化。
关闭选项卡,然后在“创建文件”下打开“新建项目”,并使用适当的名称保存数据。
接下来,打开分析向导,选择平衡,单击下一步。在此之后以各自的浓度进料。
最后,单击下一步,可以成功执行动力学参数的计算。
 


D:\ Reformatting \ 2019-12-30 \ 1902759--1277 Shalini Agarwal 797310 \ Figs jpg \ fig8.jpg


图8.固定化EhP3的细胞骨架蛋白的SPR分析。A. 表面等离子共振用于确定EhP3与细胞骨架蛋白(如Filamin,Actophorin ,Profilin A和B,Twinfilin 和EhCoactosin)之间的相互作用。EhP3通过酰胺偶联共价固定在金传感器上。此处显示的是以200 nM 浓度注入的各种蛋白质的传感图。B. 基于(A)中观察到的感应图,我们描述了此卡通模型中的相互作用,其中只有EhCoactosin 显示与EhP3结合。其他蛋白质不能与固定化的蛋白质EhP3结合。C. 此处显示浓度不断增加的EhCoactosin 注射液的传感图。使用平衡结合曲线推导亲和常数KD。重组His 6 -Ecocoactosin 的浓度显示在结合曲线旁边。






笔记


 


用于SPR研究的蛋白质的理想缓冲液是HEPES,但是如果蛋白质的稳定性成为问题,则应将任何其他缓冲液的浓度保持在最大50 mM。
EDC和NHS应优选新鲜制备,因为它们在溶液中不太稳定。如果准备量较大,应将其存储为300μ升在-20℃等份如作为解冻长下一次。但是,不建议在解冻一次后将其保存。
在乙酸盐缓冲液中配体的制备应始终新鲜进行。此外,在整个固定过程中应保持相同的乙酸盐缓冲液原液。
如果蛋白质不同,偶联缓冲液的pH值可能会有所不同。可以使用通用方程式确定偶联缓冲液的pH:pH(缓冲液)= pI (配体蛋白)-0.5。pI 是蛋白质的等电pH,在该pH下其所有负电荷都等于其所有正电荷,从而使其呈电中性。从理论上可以计算出任何具有已知氨基酸组成的蛋白质。
SPR角的偏移与结合材料的数量之间存在线性关系。120 m°的变化代表〜1 ng / mm 2的表面蛋白质质量的变化。
本机不建议使用大多数有机溶剂,请避免在任何样品前处理中使用这些有机溶剂。
具有最低折射率的醋酸盐缓冲液可提供最小的SPR角,因此是固定化过程的最佳缓冲液。乙醇胺溶液记录了最大的SPR角。
对于任何新的蛋白质-蛋白质相互作用,通常可以参考这些蛋白质的可用数据(如果有)来确定浓度。否则,可以先采用多种浓度,例如50 nM - 500 nM。如果在该范围没有给出信息,一个可以增加范围高达2 μ 中号。根据这些反应曲线,可以为特定的蛋白质组选择理想的浓度。
仪器维护:
由于浸油不可避免地溢出,应定期清洁半圆筒。如图2所示,应小心卸下M3X3螺钉,并用乙醇或在超声波水浴中清洗半圆筒。要干燥设备,必须仅使用镜头纸。清洁和干燥后,必须放置半圆筒并将其拧回到滑块上。
为了避免溶液从比色皿中蒸发,请用对位膜覆盖。
始终用蒸馏水清洁仪器。使用任何类型的缓冲剂都会导致盐沉积,从而使涂层无用。
注射泵的密封件应每年更换一次,以使仪器达到最佳性能。
 


 


菜谱


 


11-MUA解决方案
11毫克巯基十一烷酸


50毫升乙醇


过滤器对溶液进行灭菌以获得最佳结果


400 mM EDC(2毫升)
153.4毫克EDC


2毫升去离子水


100 mM NHS
23毫克NHS


2毫升去离子水


1 M乙醇胺
盐0.97克


9毫升的去离子水


使用HCl将其pH值调整为8.5


最后补足10毫升


1 M醋酸盐缓冲液pH 4.5
盐1.36克


9毫升的去离子水


用乙酸调节pH至4.5


最后,将体积补足至10 ml(Abdul Rehman 等人,2013)


1 M HEPES缓冲液pH 7.5
盐26.03克


80毫升的去离子水


用HCl调节pH值至7.5


最后补足到100 ml


5 M氯化钠
盐29.22克


100毫升的去离子水


1 M氢氧化钠
0.4克NaOH


10毫升的去离子水


10%吐温20
用去离子水配制


缓冲区1(耦合缓冲区)
用去离子水将0.5 ml醋酸盐缓冲液稀释至50 ml溶液


缓冲液2(关联缓冲液)(1 L)
10 ml 1 M HEPES缓冲液pH 7.5


从其储备溶液中提取30 ml NaCl


0.5毫升10%吐温20


用去离子水将最终体积补足至1 L


筛选和使用


缓冲区3(再生缓冲区)
从NaOH储备溶液中取出2.5毫升


用去离子水补足至50毫升


P rotein解决方案
将mg / ml蛋白质浓度转换为摩尔浓度。使用以下公式:
μ 中号蛋白=(μ 克/ 微升的蛋白质)/ (在Da MW)


接下来,稀释缓冲液2中的蛋白质储备液,以制备100μM 的工作储备液。
使用以下计算在缓冲液2(关联缓冲液)中准备稀释液:
样品名称蛋白质工作储备液的体积缓冲液2的体积                           


稀释1(1 μ 中号)1 微升99 微升                           


稀释液2(2 μ 中号)2 微升98 微升                           


稀释3(4 μ 中号)4 微升96 微升                           


稀释4(8 μ 中号)8 微升92 微升                           


稀释5(10 μ 中号)10 微升90 微升                           


当对一种蛋白质使用不同的蛋白质时,请对所有蛋白质使用相同的稀释液(在缓冲液2中稀释)。将所有用作工作储备液的蛋白质稀释1μM 。取20 升这种储备液,并添加80 升缓冲液2以制备200 nM 溶液。
蛋白质名称浓度             


分析物1(例如:EhCoactosin )200 nM             


分析物2(例如:Profilin A)200 nM             


分析物3(例如:Profilin B)200 nM             


分析物4(例如Filamin)200 nM             


分析物5(例如:Twinfilin )200 nM             


分析物6(例如:Actophorin )200 nM             


 


致谢


 


我们感谢高级仪器研究机构,JNU和Manu Vashistha 进行SPR研究。我们还要感谢Alok Bhattacharya教授(印度新德里的Ashoka大学)和Samudrala Gourinath 教授(印度新德里的Jawaharlal Nehru大学)在研究过程中为我们提供了必要的帮助。SA感谢科学技术部颁发的DST激励教师奖和补助金。PPR感谢科学和工业研究理事会(CSIR)的SRF奖学金。


 


 


利益争夺


 


作者宣称不存在任何竞争利益。


 


参考文献


 


Abdul Rehman,SA,Verma,V.,Mazumder ,M.,Dhar,SK和Gourinath ,S.(2013年)。幽门螺杆菌primase的C末端结构域的解旋酶结合的晶体结构和模式。Ĵ 细菌学195(12):2826至2838年。
Agarwal,S.,Anand,G.,Sharma,S.,Parimita Rath ,P.,Gourinath ,S.和Bhattacharya,A.(2019)。EhP3,14-3-3家族蛋白参与的在肌动蛋白重组和吞噬同源阿米巴。PLoS Pathog 15(5):e1007789。
戴维斯,TM和威尔逊,WD (2000年)。确定小分子的折射率增量,以校正表面等离子体共振数据。肛门生物化学284:348-353。
Kumar,N.,Somlata ,Mazumder ,M.,Dutta,P.,Maiti ,S.和Gourinath ,S.(2014)。EhCoactosin 稳定原生质虫Entamoeba histolytica中的肌动蛋白丝。PLoS Pathog 10(9):e1004362。
Nguyen,HH,Park,J.,Kang,S. and Kim,M.(2015年)。表面等离子体共振:一种用于生物传感器的通用技术。传感器(巴塞尔)15(5):10481-10510。
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引用:Rath, P. P., Anand, G. and Agarwal, S. (2020). Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT. Bio-protocol 10(4): e3519. DOI: 10.21769/BioProtoc.3519.
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