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In vitro Microtubule Binding Assay and Dissociation Constant Estimation
体外微管结合实验和解离常数估算   

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Abstract

Microtubules (MTs) support an astonishing set of versatile cellular functions ranging from cell division, vesicle transport, and cell and tissue morphogenesis in various organisms. This versatility is in large mediated by MT-associated proteins (MAPs). The neuronal MAP Tau, for example, is stabilizing MTs in axons of the vertebrate nervous system and thus provides the basis for enduring axonal transport and the long life span of neurons (Mandelkow et al., 1994). Tau has been shown to bind to MTs directly in vitro and also to promote their nucleation from α-/β-tubulin subunits (Goode et al., 1994). Recently, we identified a plant-specific protein family called “companion of cellulose synthase” (CC), which was shown to bind MTs and enhance dynamics of the cortical MT array in plant cells under salt stress (Endler et al., 2015). The CCs were therefore hypothesized to help plant cells cope with stress conditions and thereby maintain biomass production under adverse growth conditions. Here, we provide detailed experimental information on in vitro MT binding assays, which allow assessing whether a protein of interest is binding to MTs. The assay utilizes the high molecular weight of MTs in a spin down approach and enables the determination of the dissociation constant Kd, a measure for the protein’s binding strength to MTs.

Materials and Reagents

  1. Tubes, with Snap-On Cap, Polypropylene (1.5 ml, 11 x 38 mm) Natural (Beckman Coulter, catalog number: 357448 )
    Note: Any ultracentrifuge tubes can be used. Make sure the volume of the tubes does not exceed 1.5 to 2 ml. Otherwise handling of the assay is unfeasible.
  2. PIPES (Sigma-Aldrich, catalog number: P6757 )
  3. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  4. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  5. Ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  6. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  8. Paclitaxel (Taxol) (Sigma-Aldrich, catalog number: T7402 )
  9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855 )
  10. Guanosine 5’-triphosphate sodium salt hydrate (GTP) (Sigma-Aldrich, catalog number: G8877 )
  11. Tubulin protein, bovine, lyophilized (> 99% pure) (Cytoskeleton Inc., catalog number: TL238 ) or Tubulin protein (> 99% pure): porcine brain (Cytoskeleton Inc., catalog number: TL240 )
  12. Microtubule-associated protein rich fraction: bovine brain (Cytoskeleton Inc., catalog number: MAPF )
  13. NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well (Thermo Fisher Scientific, catalog number: NP0323PK2 )
    Note: Any other 1D protein gel able to separate tubulin and the protein of interest works as well.
  14. Brilliant Blue G (Sigma-Aldrich, catalog number: 27815 )
  15. Aluminum sulfate-(14-18)-hydrate (Sigma-Aldrich, catalog number: 368458 )
  16. 85% Orthophosphoric acid (Sigma-Aldrich, catalog number: 345245 )
  17. Sodium dodecyl sulfate (Sigma-Aldrich, catalog number: L3771 )
  18. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  19. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  20. Brinkley buffer 1980 (BRB80) (see Recipes)
  21. BRB80 Cushion buffer (see Recipes)
  22. Taxol stocks (see Recipes)
  23. GTP stocks (see Recipes)
  24. Tubulin stocks (see Recipes)
  25. MAP fraction stocks (see Recipes)
  26. BSA stocks (see Recipes)
  27. Colloidal Coomassie stain (see Recipes)
  28. Laemmli buffer (see Recipes)

Equipment

  1. Optima MAX-XP Ultracentrifuge (Beckman Coulter, catalog number: 393315 )
  2. TLA-55 Rotor Package, Fixed-Angle (45° Angle), Aluminium (Beckman Coulter, catalog number: 366725 )
    Note: Any combination of ultracentrifuge and rotor can be used.
  3. BIO RAD ChemiDOC MP Imaging System (Bio-Rad Laboratories, catalog number: 1708280 )
    Note: A normal document scanner can be used to scan protein gels instead. The scanner needs to be able to export scans in an uncompressed format.
  4. SDS-PAGE system to separate protein in 1D
  5. Vortex mixer

Software

  1. ImageJ (Fiji)
  2. Spreadsheet software (e.g., Microsoft Excel)
  3. GraphPad Prism Version 6.01 (or higher) (GraphPad Software Inc)

Procedure

  1. MT polymerization
    1. Defrost one taxol aliquot at room temperature (RT) (10 µl of 10 mM taxol in DMSO) and mix it with 990 µl of BRB80 (BRB80-T) (final taxol concentration = 100 µM). Keep at RT.
    2. Defrost one tubulin aliquot (10 µl of 4 mg/ml tubulin) in an RT water bath until thawed. Immediately transfer aliquot to ice.
    3. Add 1 µl of BRB80 Cushion buffer to the tubulin aliquot and incubate for 20 min at 37 °C in a water bath to polymerize MTs. We advise to use a water bath for uniform heating of the sample.
    4. After incubation add 100 µl of BRB80-T to the MTs to stabilize them (final tubulin concentration = approx. 4 µM). Keep at RT from now on.

  2. MT binding assay
    Note: The assay relies on the fact that MTs, because of their large molecular weight, pellet when spun at 100,000 x g. If a protein of interest binds to MTs, it will thus also be found in the pellet. For the assay to provide reliable results, it is necessary that the test protein is stable under the conditions used during centrifugation (check various buffers if that is not the case). We recommend testing the stability of your protein by running a centrifugation of your protein in buffer solution without adding MTs. This test ascertains that the protein is not (or only negligibly) found in the pellet in absence of MTs (please also refer to Figure 1). In general, the salt concentration in the assay should be kept minimal as high salt concentrations interfere with MT binding. In our hands, the interaction between the MTs and the CC proteins was affected at NaCl concentrations above approx. 60 mM.
    1. Prepare 7 reactions as described in Table 1. BSA is used as a negative control as it does not bind to MTs. A mixture of different MT binding proteins (MAPF) serves as positive control. The concentration of the test protein should be high enough to add a minimum of 5 µg of protein to the assay. Fill up to a volume of 50 µl using BRB80-T. For practical guidelines on protein expression in E. coli, please refer to Sivashanmugam et al. (2009) and Rosano and Ceccarelli (2014).

      Table 1. Experimental setup for the microtubule binding assay


    2. Incubate the reactions for 30 min at RT.
    3. Defrost one aliquot of taxol and add 990 µl of BRB80 Cushion buffer (BRB80-CT). Mix gently by pipetting up and down without introducing air bubbles. If air bubbles are introduced, spin the buffer 30 sec at 10,000 x g.
    4. Prepare 7 ultracentrifuge tubes and add 100 µl of BRB80-CT to each tube. Again, avoid introducing air bubbles to the buffer.
    5. Place the reactions as outlined in Table 1 gently on top of the BRB80-CT without mixing the 2 solutions. By spinning the reactions through the cushion buffer, the separation of unbound MAPs from the microtubules is enhanced.
    6. Centrifuge for 30 min at 100,000 x g, 23 °C.
    7. After centrifuging, label the side of the tube facing away from the rotor centre with a pen. This is where the pellet should be found (the pellet may not be visible).
    8. Note that after centrifugation the phase separation between cushion and reaction is not visible anymore. Carefully remove 30 µl from the topmost part of the solution. This is the soluble fraction of unbound protein (see Figure 1). Do not discard it!
    9. Carefully remove and discard the rest of the solution using a pipette. Avoid touching the area marked previously to not disturb the MT pellet. Try not to leave any solution inside of the tube as this will dilute the pellet fraction. The pellet will be located at the tube side and not at the actual bottom of the tube.
    10. Add 60 µl 1x Laemmli buffer (Laemmli, 1970) directly to the pellet. Mix and resuspend by vortexing.
    11. Add 6 µl of 5x Laemmli buffer to the supernatant fraction. Mix by vortexing.
    12. Separate 10 µl of each fraction by SDS PAGE and stain the gels with colloidal Coomassie stain (Dyballa and Metzger, 2009) and image the gels. See Figure 1 for representative results.


      Figure 1. Representative data showing a MT binding assay. The test protein is the His-tagged, cytosolic domain of CC1 (CC1∆C223). A MAP fraction (MAPF) was used as the positive and BSA was used as the negative control, respectively. In comparison to the control without MTs (second last lane), CC1∆C223 was significantly enriched in the pellet fraction when MTs were present (last lane). The figure was taken from Endler et al. (2015).

  3. Dissociation constant (Kd) estimation of an MT binding protein by gel densitometry
    Note: For this assay one assumes that the underlying binding mechanism of the protein to MTs follows a one-to-one stoichiometry, i.e. one molecule of protein binds to one α-/β-tubulin subunit. If this is not the case for the protein of interest, the calculated ratio of protein bound to tubulin will eventually exceed 1:1 and the presented method is, in this case, not valid. Furthermore, the reliability of the assay depends on the MTs being stable throughout the whole experiment. If your protein shows depolymerizing or severing activity on MTs, this assay is also not appropriate (the more protein one adds, the more MTs will depolymerize or be severed, respectively).
    1. Polymerize MTs (as described in part A).
    2. Prepare 6 different dilutions of the test protein with the same buffer conditions, not exceeding a volume of 30 µl. For example, for dilutions with a total volume of V = 10 µl, use x µl of test protein and add 10-x µl of buffer to maintain a stable salt concentration over all dilutions.
      Note: If a volume of 20 µl is exceeded taxol may be added to a concentration of 100 µM to prevent MT depolymerisation.
    3. Replicate the sample with the highest protein concentration as a control.
    4. Prepare 8 reactions as described in Table 2. It is essential that the final assays have the same conditions (see step C2).
      Note: To estimate the Kd accurately, the reaction has to reach saturation, i.e., an increase in protein concentration does not lead to an increase in MT binding. Firstly, we recommend performing several spin down assays with variable concentrations of the test protein to find the point of saturation. After determination of the saturation point, it is important to collect enough data points below the saturation point to accurately fit the curve and calculate the Kd. It is good practice, to repeat the experiment (at least) 3 times to acquire enough data points for robust analysis.

      Table 2. Experimental setup for the Kd estimation


    5. Incubate the reactions for 30 min at RT.
    6. Defrost one aliquot of taxol at RT and add 990 µl of BRB80 Cushion buffer. Mix gently by pipetting up and down without introducing air bubbles. If air bubbles are introduced, spin the buffer 30 sec at 10,000 x g.
    7. Prepare 8 ultracentrifuge tubes and add 100 µl of BRB80-CT to each tube. Again, avoid introducing air bubbles to the buffer.
    8. Place the reactions as outlined in Table 2 gently on top of the BRB80-CT without mixing the 2 solutions. By spinning the reactions through the cushion buffer one enhances separation of unbound MAPs from the microtubules.
    9. Centrifuge for 30 min at 100,000 x g, 23 °C.
    10. After centrifuging, label the side of the tube facing away from the rotor centre with a pen. This is where the pellet should be found (the pellet may not be visible).
    11. Note that after centrifugation the phase separation between cushion and reaction is not visible anymore. Carefully remove 30 µl from the topmost part of the solution. This is the soluble fraction of unbound protein (see Figure 1). Do not discard it!
    12. Carefully remove and discard the rest of the solution using a pipette. Avoid touching the area marked previously to not disturb the MT pellet. Try not to leave any solution inside of the tube as this will dilute the pellet fraction. The pellet will be located at the tube side and not at the actual bottom of the tube.
    13. Add 60 µl 1x Laemmli buffer (Laemmli, 1970) directly to the pellet. Mix and resuspend by vortexing.
    14. Add 6 µl of 5x Laemmli buffer to the supernatant fraction. Mix by vortexing.
    15. Load 10 µl of each fraction on an SDS gel in the order shown in Figure 2A (representative results of supernatant fractions). Also load a BSA standard (5 µg) on each gel to correct for differences in staining between each gel (right-most column in Figure 2A).
    16. Separate the fractions by SDS page, stain the gels with colloidal Coomassie stain (Dyballa et al., 2009) and image/scan them. Save the image files in an uncompressed file format.

Representative data and analysis

  1. Open the scanned gel with Fiji.
  2. Choose the “Rectangular Selections tool” and draw a rectangle around the first lane. The outline of the rectangle should be wide enough to cover each lane on your gel. Press “1” on the keyboard.
  3. A “1” will appear in the first selection. Use the mouse to click and hold inside of the first selection and drag it over to the second lane. Press “2” on your keyboard.
  4. A “2” will appear in the second selection. Repeat step 3 until you selected each lane on the gel with a separate rectangle. Do not forget to press “2” on the keyboard once you dropped the selection on a new lane. See Figure 2B for representative data with all lanes selected.
  5. Press “3” on the keyboard. Fiji will open a new window with a profile plot of each selected lane (see Figure 2C; to save space, only 4 lanes out of 9 are shown). This plot shows the relative density of each band in a lane. The lanes are arranged from top (lane 1; MT control) to bottom (lane 9; BSA standard).
  6. Choose the “Straight line tool” and enclose the peak right above the background signal (see Figure 2D). Make sure to enclose the peaks otherwise the next step will fail.
  7. Choose the “Wand tool” and click in each peak. Begin in the upper left corner (MT band from the MT control sample) and go through the all plots until you reach lane 9 (BSA standard band). A result window will open showing you the area of each peak (see Figure 2E).
  8. Once all peaks are analysed using the “Wand tool”, click “Analyze > Gels > Label Peaks” in Fiji. Fiji will calculate the percentage of the size of each individual peak based on the total size of all peaks in the plot (see Figure 2F).


    Figure 2. Workflow showing the analysis of a representative Coomassie stained gel of supernatant fractions to estimate the Kd of a MT-binding protein. A. Coomassie-stained gel showing the supernatant fraction of 8 independent MT binding experiments. Five µg of BSA is loaded on the gel as a standard (last lane). The His-tagged, cytosolic domain of CC1 (CC1∆C223) was used in the assay. Concentrations of CC1∆C223 are indicated. The molecular weights of tubulin, CC1∆C223 and BSA are indicated by arrowheads. B to F. Workflow of operations performed in Fiji to measure the peak area of the protein bands in a profile plot as described in section C, Data analysis.

  9. Copy the data of the pellet and supernatant gels into a spreadsheet program (e.g. Microsoft Excel). Figure 3A and B show representative data to illustrate the following calculations.
  10. Calculate the relative density of each peak in comparison to the BSA standard on the gel (see Figure 3A).
  11. If the test protein is also found in the pellet in absence of MTs (test protein control, see Table 2, lane 8, and Figure 1A), we recommend quantifying it to use it for corrections later on (termed “error fraction”, see Figure 3A, red box).
  12. Calculate the total protein being present in each experiment by adding the values of the pellet band and the supernatant band for each protein concentration being tested (see Figure 3C, number 1).
  13. Correct the total protein by the error fraction.
    Note: This is a simplified approach, because it assumes that the error is always the same independent from the protein concentration in the assay (see Figure 3C, number 1).
  14. Calculate the percentage of protein bound relative to MTs using the equation highlighted in Figure 3C (number 2). If your protein alone did not migrate to the pellet, you do not have to subtract the error.
  15. Calculate the concentration of the test protein in the soluble fractions using the equation highlighted in Figure 3C (number 3). The protein found in the error fraction is supposed to be non-functional. Because of this, we did not add the error fraction on top of the soluble fraction of each experiment.


    Figure 3. Representative data showing the calculations done on a data set obtained from 2 representative gels containing the pellet and supernatant fraction of 8 independent MT binding experiments as shown in Figure 2A. A. Calculation of relative peak areas in relation to the BSA standard on each gel. In case the protein of interest is also found in the pellet in absence of MTs, we recommend calculating the error fraction by normalizing the amount of protein found in the pellet by the total amount of protein in the experiment (see red boxes). B. Calculation of the total amount of protein, the ratio of protein bound to MTs and the amount of protein in solution. C. Equations used to calculate the values in B. Small boxes in A and B indicate exemplary values used in the equations. Color-coding: Equation 1-green, Equation 2-orange, Equation 3-blue.

  16. Open GraphPad Prism and create a new XY project and choose the “Binding > Saturation binding, specific binding only” model.
  17. Copy the values of “% bound protein to microtubules” into the Y-column (see Figure 4A).
  18. Copy the values of “protein in solution (µM)” into the X-column (see Figure 4A).
  19. Press “Analyze” (highlighted in Figure 4A).
  20. Choose “Nonlinear regression (curve fit)”.
  21. The parameter options will open. Choose “Binding > Saturation”, “One site > Specific binding”.
  22. You will find your Kd and other statistical data (R2, confidence intervals etc.) in the “Results” tab.
  23. In the “Graphs” tab you find a graph displaying the fitted curve and your data points. Figure 4B shows a graph of the representative data used in this protocol.


    Figure 4. Analysis performed in GraphPad Prism. A. Analysis window of GraphPad Prism showing the data as calculated in Figure 3B. The “Analysis” button is highlighted. B. Representative curve obtained after fitting a saturation binding curve (see box) to the data shown in Figure 3B and Figure 4A.

Notes


  1. If the test protein is not stable in BRB80 buffer, one can exchange BRB80 to other buffers as long as taxol remains in the buffer to stabilize the MTs. An example of a representative experiment is shown in Figure 5. The same test protein as shown before was used but we replaced BRB80-T with 50 mM TRIS-HCl (pH 6.9), 100 µM taxol to stabilize MTs after polymerization (see section A, step 4). Under these conditions, no protein is detected in the pellet fraction in absence of MTs (lane 1). On the other hand, an increased abundance of tubulin in the supernatant is visible but this is typically not affecting the outcome of the assay as long as the amount of tubulin found in the supernatant is constant across all experiments.
  2. Keep in mind that high salt concentrations (> 50 mM, see above) can interfere with the binding of the test protein to MTs. High calcium concentrations (>1 mM) (O'Brien et al., 1997) were shown to depolymerize MTs. The pH of the buffer should be in a physiological range of approx. 6.0-8.5.
  3. If the test protein has the same molecular weight as tubulin (approx. 50 kDa) and they can thus not be separated by SDS-PAGE, it is possible to detect the protein and tubulin specifically in a western blot. In this case, we recommend using an antibody against the purification tag of the test protein and a specific antibody against α- or β-tubulin. The data analysis shown here is also working for membranes obtained via Western blotting.


    Figure 5. Representative data showing a MT binding assay.
    The test protein is the His-tagged, cytosolic domain of CC1 (CC1∆C223). In comparison to Figure 1A, BRB80-T was exchanged to 50 mM TRIS-HCl (pH 6.9), 100 µM taxol. Note that no protein is found in the pellet in absence of MTs because of enhanced protein stability. Vertical black lines denote spacing between two different gels.

Recipes

  1. Brinkley buffer 1980 (BRB80)
    80 mM PIPES, adjusted to pH 6.9 using KOH
    2 mM MgCl2
    0.5 mM EGTA
  2. BRB80 Cushion buffer
    80 mM PIPES, adjusted to pH 6.9 using KOH
    1 mM MgCl2
    1 mM EGTA
    60% glycerol
  3. Taxol stocks
    10 mM Taxol in DMSO
    Split into 10 µl aliquots
    Stored at -20 °C
  4. GTP stocks
    20 mM in BRB80
    Split into 12.5 µl aliquots
    Stored at -20 °C
  5. Tubulin stocks
    Keep on ice (!)
    Mix 247.5 µl ice cold BRB80 with 12.5 µl GTP stock (= 250 µl BRB80 + 1 mM GTP)
    Reconstitute 1 mg Tubulin with 250 µl ice cold BRB80 + 1 mM GTP (concentration = 4 mg/ml)
    Gently flick the tube until tubulin is dissolved
    Split into 10 µl aliquots on ice
    Snap freeze in liquid nitrogen
    Stored at -80 °C
  6. MAP fraction stocks
    Reconstitute 100 µg of the MAP fraction in 100 µl ice cold Milli-Q water (final MAP concentration of 1 mg/ml)
    Split into 11 µl aliquots on ice
    Snap freeze in liquid nitrogen
    Stored at -80 °C
  7. BSA stocks
    Prepare a BSA solution in ice cold BRB80 with a concentration of 2.5 mg/ml
    Split into 5 µl aliquots on ice
    Snap freeze in liquid nitrogen
    Stored at -80 °C
  8. Colloidal Coomassie stain
    Please follow the protocol of Dyballa and Metzger (2009)
  9. Laemmli buffer
    60 mM Tris-HCl (pH 6.8)
    2% SDS
    10% glycerol
    5% β-mercaptoethanol
    0.01% bromophenol blue

Acknowledgments

This protocol was adapted and modified from Goode et al. (1994), Gustke et al. (1994) and the datasheet from “Microtubule Binding Protein Spin-down Assay Kit” sold by Cytoskeleton, Inc. (http://www.cytoskeleton.com/pdf-storage/datasheets/bk029.pdf).
CK was funded from an IMPRS fellowship via the Max Planck Society and a Melbourne International Research Scholarship via the University of Melbourne. SP was supported by an R@MAP Professor position at UoM. Part of the research was funded through the DFG grant PE1642/6-1 and the ARC grant DP150103495.

References

  1. Dyballa, N. and Metzger, S. (2009). Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp (30).
  2. Endler, A., Kesten, C., Schneider, R., Zhang, Y., Ivakov, A., Froehlich, A., Funke, N. and Persson, S. (2015). A mechanism for sustained cellulose synthesis during salt stress. Cell 162(6): 1353-1364.
  3. Goode, B. L. and Feinstein, S. C. (1994). Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau. J Cell Biol 124(5): 769-782.
  4. Gustke, N., Trinczek, B., Biernat, J., Mandelkow, E. M. and Mandelkow, E. (1994). Domains of tau protein and interactions with microtubules. Biochemistry 33(32): 9511-9522.
  5. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
  6. Mandelkow, E. and Mandelkow, E. M. (1995). Microtubules and microtubule-associated proteins. Curr Opin Cell Biol 7(1): 72-81.
  7. O’Brien, E. T., Salmon, E. D. and Erickson, H. P. (1997). How calcium causes microtubule depolymerization. Cell Motility and the Cytoskeleton 36(2): 125-135.
  8. Rosano, G. L. and Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5: 172.
  9. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  10. Schindelin, J., Rueden, C. T., Hiner, M. C. and Eliceiri, K. W. (2015). The ImageJ ecosystem: An open platform for biomedical image analysis. Mol Reprod Dev 82(7-8): 518-529.
  11. Sivashanmugam, A., Murray, V., Cui, C., Zhang, Y., Wang, J. and Li, Q. (2009). Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18(5): 936-948.

简介

微管(MT)支持惊人的一套多功能细胞功能,从细胞分裂,囊泡运输,以及细胞和组织形态发生在各种生物体。这种多功能性大量由MT相关蛋白(MAP)介导。例如,神经元MAP Tau是稳定脊椎动物神经系统轴突中的MT,因此为持久的轴突运输和神经元的长寿命提供了基础(Mandelkow等人,1994)。 Tau已经显示在体外直接结合MT,并且促进它们从α-/β-微管蛋白亚基的成核(Goode等人,1994)。最近,我们鉴定了称为"纤维素合酶的伴侣"(CC)的植物特异性蛋白质家族,其被证明结合MT并在盐胁迫下增强植物细胞中皮质MT阵列的动力学(Endler等,2015)。因此,CC被假定为帮助植物细胞应对胁迫条件,从而在不利生长条件下维持生物量生产。在这里,我们提供详细的实验信息在体外 MT结合测定,其允许评估感兴趣的蛋白质是否绑定到MT。该测定法在自旋向下方法中利用高分子量的MT,并且使得能够测定解离常数K d sub,其是蛋白质与MT的结合强度的量度。

材料和试剂

  1. 带有卡扣帽的管,聚丙烯(1.5ml,11×38mm)Natural(Beckman Coulter,目录号:357448)
    注意:可以使用任何超速离心管。确保管的体积不超过1.5到2毫升。否则,测定的处理是不可行的。
  2. PIPES(Sigma-Aldrich,目录号:P6757)
  3. (Sigma-Aldrich,目录号:T1503)
  4. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  5. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  6. 甘油(Sigma-Aldrich,目录号:G5516)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  8. 紫杉醇(Taxol)(Sigma-Aldrich,目录号:T7402)
  9. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:276855)
  10. 鸟苷5'-三磷酸钠盐水合物(GTP)(Sigma-Aldrich,目录号:G8877)
  11. 微球蛋白,牛,冻干(> 99%纯)(Cytoskeleton Inc.,目录号:TL238)或微管蛋白(> 99%纯):猪脑(Cytoskeleton Inc.,目录号:TL240)
  12. 微管相关蛋白富集部分:牛脑(Cytoskeleton Inc.,目录号:MAPF)
  13. NuPAGE Novex 4-12%Bis-Tris蛋白质凝胶,1.0mm,15孔(Thermo Fisher Scientific,目录号:NP0323PK2)
    注意: 任何其他能够分离微管蛋白和目标蛋白的一维蛋白凝胶都可以工作。
  14. Brilliant Blue G(Sigma-Aldrich,目录号:27815)
  15. 硫酸铝 - (14-18) - 水合物(Sigma-Aldrich,目录号:368458)
  16. 85%正磷酸(Sigma-Aldrich,目录号:345245)
  17. 十二烷基硫酸钠(Sigma-Aldrich,目录号:L3771)
  18. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  19. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  20. Brinkley缓冲液1980(BRB80)(参见配方)
  21. BRB80缓冲垫(见配方)
  22. 紫杉醇原料(见配方)
  23. GTP股票(见配方)
  24. 微管坯料(见配方)
  25. MAP级分股(见配方)
  26. BSA股票(见配方)
  27. 胶体考马斯染色(参见配方)
  28. Laemmli缓冲区(请参阅配方)

设备

  1. Optima MAX-XP超速离心机(Beckman Coulter,目录号:393315)
  2. TLA-55转子组件,固定角(45°角),铝(Beckman Coulter,目录号:366725)
    注意:可以使用超速离心机和转子的任何组合。
  3. BIO RAD ChemiDOC MP成像系统(Bio-Rad Laboratories,目录号:1708280) 注意:正常的文档扫描仪可以用来扫描蛋白质凝胶。扫描仪需要能够以未压缩的格式导出扫描。
  4. SDS-PAGE系统分离1D中的蛋白质
  5. 涡流搅拌器

软件

  1. ImageJ(斐济)
  2. 电子表格软件(例如 Microsoft Excel)
  3. GraphPad Prism版本6.01(或更高版本)(GraphPad Software Inc)

程序

  1. MT聚合
    1. 在室温(RT)除霜一个紫杉醇等分试样(10μl的10mM紫杉醇 ?在DMSO中),并与990μlBRB80(BRB80-T)(最终紫杉酚 浓度=100μM)。保持在室温。
    2. 解冻一个微管蛋白等分试样 (10μl的4mg/ml微管蛋白)在RT水浴中孵育直至解冻。立即 将等分试样转移到冰上。
    3. 加入1微升BRB80缓冲缓冲液 ?微管蛋白等分试样并在37℃下在水浴中孵育20分钟 聚合MT。我们建议使用水浴均匀加热 ?样品
    4. 孵育后加入100微升的BRB80-T到MT 稳定它们(最终微管蛋白浓度=约4μM)。保持在室温 从现在开始。

  2. MT结合测定
    注意:该测定依赖于以下事实:MTs,因为它们的大分子量,当在100,000xg下纺丝时,沉淀。如果感兴趣的蛋白质与MT结合,则它也将在沉淀中发现。对于提供可靠结果的测定,需要测试蛋白在离心期间使用的条件下是稳定的(如果不是这样,则检查多种缓冲液)。我们建议您通过在缓冲溶液中离心分离蛋白质而不添加MT来测试蛋白质的稳定性。该测试确定在不存在MT的情况下在沉淀中发现蛋白质(或仅可忽略地)(也请参见图1)。通常,测定中的盐浓度应当保持最小,因为高盐浓度干扰MT结合。在我们的手中,MT和CC蛋白之间的相互作用在NaCl浓度高于约10μM时受到影响。 60 mM。
    1. 准备如表1所述的7个反应。使用BSA作为阴性 控制,因为它不绑定到MT。不同MT结合的混合物 蛋白质(MAPF)用作阳性对照。浓度的 测试蛋白应该足够高以至少添加5μg的蛋白质 到测定。使用BRB80-T填充至50μl的体积。实用 关于蛋白质表达的指南。大肠杆菌请参阅 Sivashanmugam等人(2009年)和Rosano和Ceccarelli(2014年)。

      表1.微管结合试验的实验设置


    2. 在室温下孵育反应30分钟。
    3. 解冻一份紫杉醇,加入990μlBRB80缓冲液 (BRB80-CT)。通过上下吹吸混合轻轻混合,不引入空气 气泡。如果引入气泡,则以10,000×g 旋转缓冲液30秒
    4. 准备7超速离心管,并添加100微升的BRB80-CT到每个管。同样,避免向缓冲液中引入气泡。
    5. 将反应如表1所示轻轻地放在顶部 BRB80-CT,不混合2种溶液。通过旋转反应 通过缓冲缓冲区将未结合的MAP的分离 微管增强。
    6. 在100,000xg,23℃下离心30分钟。
    7. 离心后,标记管的侧面远离 ?转子中心用笔。这是颗粒应该被发现( 颗粒可能不可见)。
    8. 注意离心后 缓冲和反应之间的相分离不再可见。 小心地从溶液的最顶部除去30微升。这是 未结合蛋白的可溶性级分(参见图1)。不要丢弃 它!
    9. 小心地去除和丢弃其余的溶液使用 ?吸管。避免触摸以前标记的区域,以免打扰 MT颗粒。尝试不要留在管内的任何解决方案,因为这将是 ?稀释沉淀部分。颗粒将位于管侧 ?而不是在管的实际底部
    10. 加入60μl1x Laemmli缓冲液(Laemmli,1970)直接到沉淀。混合并通过涡旋重悬
    11. 向上清液部分中加入6μl5x Laemmli缓冲液。通过涡旋混合。
    12. 通过SDS PAGE分离10μl的每个部分,并用凝胶染色 ?胶体考马斯染色(Dyballa和Metzger,2009)和图像 凝胶。参见图1的代表性结果。


      图1。 显示MT结合测定的代表性数据。测试蛋白是 CC1的带His标签的胞质结构域(CC1ΔC223)。 MAP分数(MAPF) 用作阳性,BSA用作阴性对照, 分别。与没有MT的控制相比(倒数第二 泳道),CC1ΔC223在沉淀组分中显着富集 存在MT(最后一泳道)。该图取自Endler等人(2015)。

  3. 通过凝胶光密度测定MT结合蛋白的解离常数( K d )
    注意:对于该测定,假定蛋白质与MT的基本结合机制遵循一对一化学计量,即一个蛋白质分子结合一个α-/β-微管蛋白亚基。如果感兴趣的蛋白质的情况不是这样,则与微管蛋白结合的蛋白质的计算比率将最终超过1:1,并且所提出的方法在这种情况下是无效的。此外,测定的可靠性取决于MT在整个实验中是稳定的。如果你的蛋白质在MT上显示解聚或切断活性,这个测定也是不合适的(蛋白质越多,分别解聚或被切断的MT越多)。
    1. 聚合MT(如A部分所述)。
    2. 准备6种不同 用相同的缓冲液条件稀释测试蛋白质,而不是 超过30μl的体积。例如,对于总量的稀释 体积V =10μl,使用xμl的测试蛋白质并加入10-xμl缓冲液 以在所有稀释液中保持稳定的盐浓度。
      注意:如果超过20μl的体积,可以加入浓度为100μM的紫杉醇以防止MT解聚。
    3. 复制具有最高蛋白质浓度的样品作为对照
    4. 准备8个反应,如表2所述。最后的测定具有相同的条件(参见步骤C2)是重要的 注意:为了准确估计K ,反应必须达到 饱和,即蛋白质浓度的增加不会导致 MT结合的增加。首先,我们建议执行几个旋转 ?使用可变浓度的测试蛋白进行测定 饱和点。确定饱和点后,即可 ?对于在饱和点以下收集足够的数据点是很重要的 以精确拟合曲线并计算Kd。这是好的做法, 重复实验(至少)3次以获取足够的数据 分数,用于稳健分析。

      表2. 估计的实验设置


    5. 在室温下孵育反应30分钟。
    6. 在室温下解冻一份紫杉醇,加入990μlBRB80缓冲垫 缓冲。通过上下吹吸混合轻轻混合,不引入空气 气泡。如果引入气泡,则以10,000×g 旋转缓冲液30秒
    7. 准备8超速离心管,并添加100微升BRB80-CT到每个管。同样,避免向缓冲液中引入气泡。
    8. 将反应如表2所示温和地放置在顶部 BRB80-CT,不混合2种溶液。通过旋转反应 通过缓冲缓冲液增强未结合的MAP的分离 微管
    9. 在100,000xg,23℃下离心30分钟。
    10. 离心后,标记管的侧面远离 ?转子中心用笔。这是颗粒应该被发现( 颗粒可能不可见)。
    11. 注意离心后 缓冲和反应之间的相分离不再可见。 小心地从溶液的最顶部除去30微升。这是 未结合蛋白的可溶性级分(参见图1)。不要丢弃 它!
    12. 小心地去除和丢弃其余的溶液使用 ?吸管。避免触摸以前标记的区域,以免打扰 MT颗粒。尝试不要留在管内的任何解决方案,因为这将是 ?稀释沉淀部分。颗粒将位于管侧 ?而不是在管的实际底部
    13. 加入60μl1x Laemmli缓冲液(Laemmli,1970)直接到沉淀。混合并通过涡旋重悬。
    14. 向上清液部分中加入6μl5x Laemmli缓冲液。通过涡旋混合。
    15. 在SDS凝胶上按10所示的顺序加载10μl的每个组分 图2A(上清液部分的代表性结果)。也加载a ?BSA标准品(5μg),以校正染色的差异 在每个凝胶(图2A中最右侧的列)之间
    16. 分开 通过SDS PAGE分级,用胶态考马斯染色剂染色凝胶 (Dyballa等人,2009)和图像/扫描他们。将图像文件保存在 未压缩文件格式。

代表数据和分析

  1. 用斐济打开扫描的凝胶。
  2. 选择"矩形选择工具",并在第一条车道周围绘制一个矩形。矩形的轮廓应该足够宽,以覆盖凝胶上的每个泳道。在键盘上按"1"。
  3. 在第一个选择中将出现"1"。使用鼠标单击并按住第一个选择内部,并将其拖动到第二个通道。在键盘上按"2"。
  4. 在第二个选择中将出现"2"。重复步骤3,直到您选择凝胶上的每个泳道与一个单独的矩形。一旦你把一个新的车道上的选择删除,不要忘记在键盘上按"2"。有关所有通道选择的代表性数据,请参见图2B
  5. 按键盘上的"3"。斐济将打开一个新窗口,其中显示每个选定车道的轮廓图(见图2C;为了节省空间,只显示了9条车道中的4条车道)。该图显示了泳道中每个条带的相对密度。泳道从顶部(泳道1; MT对照)至底部(泳道9; BSA标准)排列。
  6. 选择"直线工具"并将峰包围在背景信号的正上方(参见图2D)。确保包围峰,否则下一步将失败。
  7. 选择"魔杖工具",然后单击每个峰。从左上角开始(从MT对照样品的MT带),并通过所有图,直到到达泳道9(BSA标准带)。将打开一个结果窗口,显示每个峰的面积(参见图2E)。
  8. 使用"Wand工具"分析所有峰后,点击"分析>凝胶>标签峰"。斐济将根据图中所有峰的总大小计算每个峰的大小百分比(见图2F)。


    图2.显示上清液级分的代表性考马斯染色凝胶的分析的工作流程,以估计MT结合的 蛋白质。A.考马斯染色的凝胶,显示8次独立的MT结合实验的上清液部分。将5μgBSA加载到凝胶上作为标准(最后一泳道)。在测定中使用CC1的His-标记的胞质结构域(CC1ΔC223)。显示了CC1ΔC223的浓度。微管蛋白,CC1ΔC223和BSA的分子量由箭头指示。 B到F.在斐济进行的操作的工作流程,用于在剖面图中测量蛋白质谱带的峰面积,如C部分,数据分析中所述。

  9. 将沉淀和上清液凝胶的数据复制到电子表格程序(例如Microsoft Excel)中。图3A和B显示了代表性数据来说明以下计算
  10. 计算每个峰相对于凝胶上BSA标准品的相对密度(见图3A)
  11. 如果在没有MT的情况下在沉淀中也发现测试蛋白质(测试蛋白质对照,参见表2,泳道8和图1A),我们推荐对其进行量化以用于后面的校正(称为"误差分数",参见图3A,红色框)。
  12. 通过加入测试的每种蛋白质浓度的沉淀带和上清液条带的值来计算每个实验中存在的总蛋白质(参见图3C,编号1)。
  13. 通过误差分数校正总蛋白。
    注意:这是一种简化的方法,因为它假设错误总是相同的,独立于测定中的蛋白质浓度(见图3C,数字1)。
  14. 使用图3C中突出显示的方程(数字2)计算相对于MT结合的蛋白质的百分比。如果你的蛋白质单独没有迁移到沉淀,你不必减去错误。
  15. 使用图3C(数字3)中突出显示的方程计算可溶性级分中测试蛋白质的浓度。在错误部分中发现的蛋白质应该是无功能的。因为这一点,我们没有在每个实验的可溶性级分的顶部添加误差分数。


    图3.代表性数据,显示对从包含8个独立的MT结合实验的沉淀和上清液部分的2个代表性凝胶获得的数据集进行的计算,如图2A所示。A.计算相对峰面积相对于每种凝胶上的BSA标准。如果在没有MT的情况下在沉淀中也发现感兴趣的蛋白质,我们建议通过在实验中将蛋白质的总量标准化沉淀中存在的蛋白质的量来计算误差分数(参见红色框)。 B.计算蛋白质总量,与MT结合的蛋白质的比例和溶液中蛋白质的量。 C.用于计算B中的值的方程式.A和B中的小方框表示在方程式中使用的示例性值。颜色编码:公式1-绿色,公式2-橙色,公式3-蓝色
  16. 打开GraphPad Prism并创建一个新的XY项目,并选择"Binding>饱和结合,特异性结合"模式
  17. 将"%结合蛋白质与微管"的值复制到Y柱中(参见图4A)。
  18. 将"溶液中的蛋白质(μM)"的值复制到X列中(参见图4A)
  19. 按"分析"(图4A中突出显示)。
  20. 选择"非线性回归(曲线拟合)"。
  21. 将打开参数选项。选择"绑定">饱和","一个站点>特异性结合"。
  22. 您将在"结果"列表中找到 K d 和其他统计数据(R2,置信区间等) "标签。
  23. 在"图表"选项卡中,可以找到一个显示拟合曲线和数据点的图表。图4B示出了在该协议中使用的代表性数据的图表

    图4.在GraphPad Prism中执行的分析 A. GraphPad Prism的分析窗口显示如图3B中计算的数据。 "分析"按钮突出显示。 B.在将饱和结合曲线(参见框)拟合到图3B和图4A所示的数据之后获得的代表性曲线

笔记


  1. 如果测试蛋白在BRB80缓冲液中不稳定,可以交换 BRB80到其他缓冲液只要紫杉醇保留在缓冲液中即可 稳定MT。代表性实验的实例如图1所示 ?图5.使用与之前相同的测试蛋白,但我们 用50mM TRIS-HCl(pH 6.9),100μM紫杉醇代替BRB80-T以稳定 ?聚合后的MT(参见A部分,步骤4)。在这些 条件下,在不存在时在沉淀级分中没有检测到蛋白质 MTs(泳道1)。另一方面,微管蛋白的丰度增加 上清液是可见的,但这通常不影响 只要发现微管蛋白的量在测定的结果 上清液在所有实验中是恒定的。
  2. 记住,高盐浓度(> 50mM,参见上文)可以 干扰测试蛋白与MT的结合。高钙 浓度(> 1mM)(O'Brien等人,1997)显示解聚MT。缓冲液的pH应该在大约1的生理范围内。 6.0-8.5。
  3. 如果测试蛋白具有与微管蛋白相同的分子量(约50μM) ?kDa),因此它们不能通过SDS-PAGE分离,有可能 在蛋白质印迹中特异性检测蛋白质和微管蛋白。在这里 病例,我们建议使用针对纯化标签的抗体 ?测试蛋白和针对α-或β-微管蛋白的特异性抗体。数据 这里显示的分析也用于通过Western获得的膜 印迹

    图5.显示MT结合测定的代表性数据。测试蛋白是CC1的His-标记的胞质结构域(CC1ΔC223)。与图1A相比,将BRB80-T更换为50mM TRIS-HCl(pH 6.9),100μM紫杉醇。注意,在不存在MT的情况下,在沉淀中没有发现蛋白质,因为增强的蛋白质稳定性。垂直黑线表示两种不同凝胶之间的间距。

食谱

  1. Brinkley缓冲液1980(BRB80)
    80mM PIPES,用KOH调节至pH6.0 2mM MgCl 2/
    0.5mM EGTA
  2. BRB80缓冲缓冲器
    80mM PIPES,用KOH调节至pH6.0 1mM MgCl 2
    1 mM EGTA
    60%甘油
  3. 紫杉醇股
    10mM紫杉醇的DMSO溶液 分成10μl等分
    储存于-20°C
  4. GTP股票
    在BRB80中为20mM
    分成12.5μl等分
    储存于-20°C
  5. 微管蛋白股票
    保持冰上(!)
    将247.5μl冰冷的BRB80与12.5μlGTP原液(=250μlBRB80 + 1mM GTP)混合
    用250μl冰冷的BRB80 + 1mM GTP(浓度= 4mg/ml)重构1mg微管蛋白。
    轻轻摇动管直到微管蛋白溶解
    在冰上分成10μl等份
    在液氮中快速冷冻
    储存于-80°C
  6. MAP分数股
    在100μl冰冷的Milli-Q水(最终MAP浓度为1mg/ml)中重构100μgMAP级分
    在冰上分成11μl等分试样
    在液氮中快速冷冻
    储存于-80°C
  7. BSA股票
    在冰冷的BRB80中制备浓度为2.5mg/ml的BSA溶液 分成5微升等分在冰上
    在液氮中快速冷冻
    储存于-80°C
  8. 胶体考马斯染色
    请按照Dyballa和Metzger(2009)的规程
  9. Laemmli缓冲区
    60 mM Tris-HCl(pH 6.8)
    2%SDS
    10%甘油 5%β-巯基乙醇 0.01%溴酚蓝

致谢

该方案由Goode等人(1994),Gustke等人(1994)和来自"Microtubule Binding Protein Spin-down Assay Kit"的数据表改编和修改,由Cytoskeleton,Inc.出售( http://www.cytoskeleton.com/pdf-存储/datasheets/bk029.pdf )。
CK通过马克斯普朗克学会的IMPRS奖学金和墨尔本大学的墨尔本国际研究奖学金资助。 SP由UMA的R @ MAP教授担任。部分研究通过DFG授予PE1642/6-1和ARC授权DP150103495资助。

参考文献

  1. Dyballa,N.和Metzger,S。(2009)。 快速和灵敏的胶体考马斯G-250染色,用于聚丙烯酰胺凝胶中的蛋白质。 J Vis Exp (30)。
  2. Endler,A.,Kesten,C.,Schneider,R.,Zhang,Y.,Ivakov,A.,Froehlich,A.,Funke,N.and Persson, 盐胁迫过程中持续纤维素合成的机制 单元 162(6):1353-1364。
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引用:Kesten, C., Schneider, R. and Persson, S. (2016). In vitro Microtubule Binding Assay and Dissociation Constant Estimation. Bio-protocol 6(6): e1759. DOI: 10.21769/BioProtoc.1759.
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