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Active Cdk5 Immunoprecipitation and Kinase Assay
活性Cdk5免疫沉淀及其激酶活性的测定   

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Abstract

Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal amounts of total protein between experimental groups. Washes are then performed to remove extraneous proteins and equilibrate the Cdk5-activator complexes in the kinase buffer. Cdk5 is then incubated with histone H1, a well-established in vitro target of Cdk5, and [γ-32P]ATP. Reactions are resolved by SDS-PAGE and transferred to membranes for visualization of H1 phosphorylation and immunoblot of immunoprecipitated Cdk5 levels. We have used this assay to establish p39 as the primary activator for Cdk5 in the oligodendroglial lineage. However, this assay is amenable to other cell lineages or tissues with appropriate adjustments made to lysis conditions.

Keywords: Kinase assay(激酶活性测定), Cdk5(Cdk5), Western blot(蛋白质印迹), Immunoblot(免疫印迹), Radiation(辐射)

Background

Although Cdk5 is typically associated with neuronal function, recent work has demonstrated that Cdk5 also regulates oligodendroglia progenitor cell (OPC) development (Tang et al., 1998; Miyamoto et al., 2007 and 2008). Cdk5 function is critical for OPC migration and differentiation, and loss of Cdk5 results in CNS hypomyelination (Miyamoto et al., 2007 and 2008; He et al., 2010; Yang et al., 2013). However, molecular mechanisms that regulate Cdk5 function in neurons and OLs remain elusive. The activity of Cdk5 is controlled by the available amounts of two activator homologs, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The defects in embryonic brain development and perinatal lethality observed in mice lacking both p35 and p39 were nearly identical to defects in the Cdk5-null mice (Ohshima et al., 1996; Ko et al., 2001), indicating that p35 and p39 are the sole activators of Cdk5 in the brain. We uncovered that in contrast to the major role of p35 in activating Cdk5 in neurons, p39 is the primary Cdk5 activator in oligodendrocytes (OLs), where p35 expression is negligible. Using this active Cdk5 immunoprecipitation and kinase assay, we demonstrated that Cdk5 activity is almost completely ablated in OLs with siRNA-mediated p39 knockdown. Previous work established the differing sensitivity of p35 and p39 to high detergent and salt concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Based on those reports, this protocol was developed to try and preserve both p35- and p39-Cdk5 complexes to measure total Cdk5 activity regardless of activator. Our work further showed that p39 is essential for OL differentiation and myelin repair, with upregulation of p35 masking the loss of p39 function during myelin development. Measuring Cdk5 activity from cells, in combination with immunoblots for Cdk5 target phosphorylation, provides a tool to identify novel regulators of Cdk5 activation.

Materials and Reagents

  1. Cell lifter (Corning, catalog number: 3008 )
  2. Corning sterile 60 mm cell culture dishes (Corning, catalog number: 3261 )
  3. Corning sterile 100 mm cell culture dishes (Corning, catalog number: 3262 )
  4. 15 ml conical tubes (Denville Scientific, catalog number: C1018-P )
  5. Micro slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4951PLUS4 )
  6. Micro cover glass (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 25X54I24901 )
  7. 1.7 ml tubes (Denville Scientific, SlipTechTM, catalog number: C19033 )
  8. 50 ml conical tubes (Denville Scientific, catalog number: 1005513 )
  9. Nitrocellulose or PVDF membrane (GE Healthcare, catalog number: RPN82D or 10600021 )
  10. Autoclaved micropipette tips (Denville Scientific, Woodpecker ReloadsTM, catalog numbers: P2102-NB , P2101-N , P2109 )
  11. X-ray film (Denville Scientific, Hyblot ES®, catalog number: E3218 )
  12. Trypan blue (Sigma-Aldrich, catalog number: 302643 )
  13. BCA Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23235 )
  14. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  15. Antibody against Cdk5 (Santa Cruz C-8) (Santa Cruz Biotechnology, catalog number: sc-173 )
  16. Antibody against Cdk5 (Cell Signaling Technology, catalog number: 2506 )
  17. Rabbit IgG antibody (Vector Laboratories, catalog number: I-1000 )
  18. Goat anti-rabbit-horseradish peroxidase (HRP) (Jackson ImmunoResearch, catalog number: 111-035-003 )
  19. 10 mM ATP (Thermo Fisher Scientific, catalog number: PV3227 )
  20. Cdk5/p25, active complex (EMD Millipore, catalog number: 14-516 )
  21. [γ-32P]ATP (PerkinElmer, catalog number: BLU002Z250UC )
  22. Histone H1 (EMD Millipore, catalog number: 14-155 )
  23. 12% polyacrylamide gel (Bio-Rad Laboratories, catalog number: 4561043 )
  24. Enhanced Chemiluminescence Reagent Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 32106 )
  25. Tris/Glycine/SDS buffer (Bio-Rad Laboratories, catalog number: 1610732 )
  26. Methanol (Sigma-Aldrich, catalog number: 34860 )
  27. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  28. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  29. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  30. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  31. Tris base (Sigma-Aldrich, catalog number: RDD008 )
  32. Concentrated HCl (Sigma-Aldrich, catalog number: 295426 )
  33. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
  34. Sodium hydroxide (NaOH) tablets (Sigma-Aldrich, catalog number: S8045 )
  35. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  36. 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich, catalog number: RDD003 )
  37. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  38. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
  39. NP-40/IGEPAL® CA-630 (Sigma-Aldrich, catalog number: I3021 )
  40. Polymethyl sulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  41. 100% ethanol (Sigma-Aldrich, catalog number: E7023 )
  42. Pepstatin (Sigma-Aldrich, catalog number: P4265 )
  43. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  44. Aprotinin (Sigma-Aldrich, catalog number: A1153 )
  45. Protein A Sepharose CL-4B (GE Healthcare, catalog number: 17-0780-01 )
  46. Pre-activated sodium orthovanadate (100 mM Na3VO4) (New England Biolabs, catalog number: P0758L )
  47. 10% BrijTM-35 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28316 )
  48. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  49. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  50. Magnesium acetate (MgOAc) (Sigma-Aldrich, catalog number: M5661 )
  51. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
  52. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  53. 1x transfer buffer (see Recipes)
  54. 10x phosphate-buffered saline (PBS) (see Recipes)
  55. 1x phosphate-buffered saline (PBS) (see Recipes)
  56. 2 M Tris-HCl (pH 7.5) (see Recipes)
  57. 50 mM Tris-HCl (pH 7.5) (see Recipes)
  58. 5 M NaCl (see Recipes)
  59. 0.5 M EDTA (pH 8.0) (see Recipes)
  60. 0.5 M EGTA (pH 8.0) (see Recipes)
  61. 1 M MOPS (pH 7.0) (see Recipes)
  62. 1 M MgCl2 (see Recipes)
  63. 1 M NaF (see Recipes)
  64. 20% NP-40 (see Recipes)
  65. 100 mM PMSF (see Recipes)
  66. 1 mg/ml pepstatin A (see Recipes)
  67. 1 mg/ml leupeptin (see Recipes)
  68. 1 mg/ml aprotinin (see Recipes)
  69. 50% slurry of Protein A Sepharose CL-4B (see Recipes)
  70. Cdk5 lysis buffer (stock) (see Recipes)
  71. Cdk5 lysis buffer (working) (see Recipes)
  72. Cdk5 kinase buffer (stock) (see Recipes)
  73. Cdk5 kinase buffer (washes) (see Recipes)
  74. Cdk5 kinase buffer (assay) (see Recipes)
  75. MOPS dilution buffer (see Recipes)
  76. 5x reaction buffer (see Recipes)
  77. 50 mM magnesium acetate buffer (MgOAc) (see Recipes)
  78. 1x phosphate-buffered saline/0.1% Tween-20 (PBS-T) (see Recipes)
  79. 5x Laemmli buffer (see Recipes)
  80. 0.2% bromophenol blue (see Recipes)

Equipment

  1. Hemacytometer (Hausser Scientific, catalog number: 3100 )
  2. PIPETMAN ClassicTM pipets (Gilson, model: P10, catalog number: F144802 )
  3. PIPETMAN ClassicTM pipets (Gilson, model: P20, catalog number: F123600 )
  4. PIPETMAN ClassicTM pipets (Gilson, model: P200, catalog number: F123601 )
  5. PIPETMAN ClassicTM pipets (Gilson, model: P1000, catalog number: F123602 )
  6. Refrigerated tabletop centrifuge for 15 ml conical tubes (Jouan, model: CT422 )
  7. Tabletop centrifuge for 1.5 ml tubes in a 4 °C cold room (Eppendorf, model: 5415 D )
  8. Inverted light microscope (Olympus, model: CK30 )
  9. Certified Geiger counter (W.B. Johnson Instruments, model: GSM-110 )
  10. Plexiglass shielding (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 6700-1812 )
  11. Phosphorimaging cassette (Thomas Scientific, catalog number: C993J84)
    Manufacture: bioWORLD, catalog number: 43121008-1 .
  12. Autoradiography cassette (Denville Scientific, catalog number: E3122 )
  13. 500 ml glass bottles (Corning, PYREX®, catalog number: 1397-500 )

Software

  1. GraphPad Prism (GraphPad Software)
  2. ImageJ (https://imagej.nih.gov/ij)

Procedure

Notes:

  1. Steps A1-A4 should be performed as quickly as possible to avoid loss of cellular proteins during washes.
  2. This protocol is designed to preserve the Cdk5/activator complex while still maintaining enough stringency to reasonably lyse the cells and remove non-specific proteins from the immunoprecipitated complex.
  3. Once [γ-32P]ATP has been added in step D3b, ALL discarded solutions MUST be disposed according to the radiation safety protocols at your institution for the remainder of this protocol.
  1. Generating cellular lysates
    1. Cell cultures should be grown to confluence with the appropriate treatment and control groups.
    2. Aspirate the media from the plate(s) and rinse the cell cultures very gently with PBS (see Recipes) twice. Remove the PBS from the last wash.
    3. Detach the cells from the culture plates using a cell lifter. Collect the detached cells by rinsing the plate with an appropriate volume of PBS (1 ml for 60 mm plates, 3 ml for 100 mm plates, etc.).
    4. Transfer the cell suspension to a 15 ml conical tube.
    5. Centrifuge the cell suspension at 3,500 x g for 5 min at 4 °C.
    6. Repeat steps A3-A5 if needed.
    7. Resuspend the cell pellets in lysis buffer (see Recipes) at 15% w/v.
    8. Incubate lysates on ice for 10 min with periodic agitation.
    9. Confirm lysis by mixing a 10 µl sample of the lysate 1:1 with trypan blue. Pipette 10 µl of this mixture onto a microscope slide and place a coverslip onto the 10 µl droplet. Good lysis will appear as many intact nuclei that are no longer contained within cells. Unlysed cells will appear as spheres with blue outlines. If the nuclei lyse, many brown fibers will be present, which can negatively impact the purity of your immunoprecipitation. Example images of unlysed cells and good lysis are presented in Figure 1.
    10. Reserve 10% of the lysate as ‘Input’, add 5x Laemmli buffer (see Recipes) to Input to a final concentration of 1x, and store samples on ice until step D9.


      Figure 1. Lysis confirmation. Left, example image of unlysed cells stained with trypan blue. Note the blue outline and slight halo around each cell. Right, example image of intact nuclei from lysed cells.

  2. Preparing sample for immunoprecipitation
    1. Centrifuge the lysate at 10,000 x g for 10 min at 4 °C.
    2. Transfer the supernatant to a fresh tube.
    3. Add 50 µl of 50% slurry of Protein A beads (or whichever type of beads you will use for the immunoprecipitation) to the lysate.
    4. Rotate or rock the mixture of beads and lysate at 4 °C for 30 min to pre-clear the lysate of proteins that non-specifically bind to the beads.
    5. Centrifuge the mixture at 3,000 x g for 2 min at 4 °C.
    6. Transfer the supernatant to a fresh tube.
    7. Quantify the concentration of protein in the lysate using standard protein quantification methods (Bradford or BCA kits, for example)
      1. Generate a standard curve of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, and 0 mg/ml bovine serum albumin (BSA) by diluting it using sterile water.
      2. Dilute the lysate (between 1:2 to 1:10 for cell lysate) so that the sample protein concentration will fall within the range of the BSA standard curve.
      3. Follow the manufacturer’s instructions to record the concentration of protein in the lysate. Take note of the R2 value as it indicates the accuracy of the protein concentration quantification (0.9 and above is desired).
      4. Take the average of three readings.

  3. Immunoprecipitating the Cdk5/activator complex
    1. Prepare two fresh 1.5 ml tubes with 1 mg of total protein from the lysate.
      Note: If one of your samples contains insufficient protein concentration, equalize the total protein amount in all samples to match the protein amounts possible from the sample with the lowest protein concentration.
    2. Add lysis buffer to bring the total volume in each tube to 500 µl.
    3. Add 10 µl (2 µg) Santa Cruz anti-Cdk5 antibody (C-8) to one of the 1.5 ml tubes prepared for each of your samples.
    4. Add 2 µg of IgG antibody to the other 1.5 ml tubes prepared for each of your samples.
    5. Rotate or rock the mixture of beads and lysate at 4 °C for 3 h.
    6. Add 50 µl of 50% slurry of Protein A beads to each tube.
    7. Rotate or rock the mixture of beads and lysate at 4 °C for 2 h.
    8. Centrifuge the tubes at 3,000 x g for 2 min at 4 °C.
    9. Remove and discard the supernatant.
    10. Add 500 µl of lysis buffer containing 0.1% NP-40.
    11. Rotate or rock the mixture of beads and lysate at 4 °C for 10 min.
    12. Repeat steps C8-C11 twice more.
    13. Centrifuge the tubes at 3,000 x g for 2 min at 4 °C.
    14. Remove and discard the supernatant.
    15. Add 500 µl of kinase buffer (see Recipes) without ATP or histone H1.
    16. Rotate or rock the mixture of beads and lysate at 4 °C for 10 min.
    17. Repeat steps C13-C16 once more (general scheme of immunoprecipitation in Figure 2).


      Figure 2. Scheme for immunoprecipitation of active Cdk5 complexes

  4. Cdk5 kinase assay
    1. Resuspend beads in 25 µl kinase buffer (with 100 ng/µl H1 and 25 µM ATP).
    2. Prepare a reaction with recombinant Cdk5/p25 for a positive control (general scheme for Cdk5 kinase assay and controls in Figure 3).
      1. Add 14.6 µl MOPS dilution buffer (see Recipes) to 1 µl Cdk5/p25 activated complex (Upstate/Millipore).
      2. Add 5 µl 5x reaction buffer (see Recipes) to a fresh 1.5 ml tube.
      3. Add 2.5 µl of histone H1 (final 100 ng/µl).
      4. Add 2.5 µl of diluted Cdk5 from step D2a to the 1.5 ml tube.
      5. Add 5 µl of sterile water.


        Figure 3. Scheme for Cdk5 kinase assay, SDS-PAGE, and detection

    3. Prepare a working solution of [γ-32P]ATP
      1. Prepare 47 µl of 25 mM magnesium acetate (see Recipes).
      2. Add [γ-32P]ATP (final concentration 0.1 µCi/µl).
      3. Add 10 mM ATP to bring the total volume to 50 µl.
    4. Add 10 µl of the 32P mix from step D3 to the positive control reaction prepared in step D2.
    5. Add 2 µl of 32P mix from step D3 to each IP reaction from step D1.
    6. Mix 25 µl of kinase buffer with 2 µl of 32P mix from step D3 as a negative control.
    7. Incubate all reactions at 30 °C for 30 min.
    8. Add 5 µl of 5x Laemmli buffer to each reaction.
    9. OPTIONAL: Store reactions at -20 °C.
    10. Resolve immediately by 12.5% SDS-PAGE.
    11. DO NOT allow the dye front to exit the bottom of the gel. This will minimize radioactive contamination of the running buffer or equipment.
    12. After resolving reactions, cut off dye at bottom (contains free 32P) and stacking gel.
    13. Transfer the resolved proteins to nitrocellulose or PVDF membrane.
    14. Expose the membrane to film or phosphorscreen at -80 °C (see example in Figure 4).


      Figure 4. Detection of Cdk5 kinase activity and protein levels in immunoprecipitates. Results of Cdk5 immunoprecipitation and kinase assay performed from cells transfected with control siRNA with empty vector (Ctrlsi + pc), p39 siRNA with empty vector (p39si + pc), p39 siRNA with p39-FLAG (p39si + p39F). Kinase buffer alone was included as a negative control. In this figure, the exogenously expressed p39-FLAG reversed the effect on Cdk5 activity caused by siRNA-mediated knockdown of endogenous p39. This research was originally published in Journal of Biological Chemistry. Bankston AN, Li W, Zhang H, Ku L, Liu G, Papa F, Zhao L, Bibb JA, Cambi F, Tiwari-Woodruff SK, Feng Y. p39, the primary activator for cyclin-dependent kinase 5 (Cdk5) in oligodendroglia, is essential for oligodendroglia differentiation and myelin repair. J Biol Chem. 2013; 288(25):18047-57. © the American Society for Biochemistry and Molecular Biology.

  5. Performing Western blot to assess immunoprecipitation
    1. Incubate the membrane for one hour with 10% non-fat milk in PBS-T (see Recipes).
    2. Discard 10% milk solution.
    3. Incubate the membrane with 2% non-fat milk in PBS-T containing a 1:1,000 dilution of anti-Cdk5 antibody (Cell Signaling Technology) at 4 °C overnight.
    4. Discard the 2% milk solution containing anti-Cdk5 antibody.
    5. Wash the membrane by incubating it in PBS-T for 10 min.
    6. Discard the PBS-T wash solution.
    7. Repeat steps E5-E6 twice more.
    8. Incubate the membrane with 2% non-fat milk in PBS-T containing a 1:5,000 dilution of HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch) at room temperature for one hour.
    9. Wash the membrane by incubating it in PBS-T for 10 min.
    10. Discard the PBS-T wash solution.
    11. Incubate the membrane with an enhanced chemiluminescence solution according to the manufacturer’s protocol.
    12. Expose the membrane to film (see example in Figure 4).

Data analysis

  1. For experimental design, the immunoprecipitation and kinase assay are performed a minimum of three times for a given experiment.
  2. Densitometric analysis is performed using ImageJ. For each sample the intensity of histone H1 phosphorylation measured in step D13 are normalized to the intensity of Cdk5 protein measured in step E12 (see Figure 5).
  3. For statistical analysis, we use GraphPad Prism software. To compare two samples, a two-tailed Student’s t-test was used. To compare three or more samples, one-way ANOVA with a post-hoc Tukey correction was used.


    Figure 5. Measurement and normalization of Cdk5 kinase activity and protein levels in ImageJ. A. Draw a box around the first lane of Cdk5 kinase activity (yellow solid box). Click Select First Lane under the Analyze Gels menu. Move the box over the second lane of Cdk5 kinase activity (yellow dashed box). Click Select Second Lane under the Analyze Gels menu. Move the box over each of the remaining lanes of Cdk5 kinase activity, clicking Select Second Lane under the Analyze Gels menu at each lane. Click Plot Lanes under the Analyze Gels menu. B. Plots will be generated for each lane. Use the line tool (indicated by bold red arrow) to draw a line across the bottom of the plot (cursor drawing line indicated by slim red arrow). Be sure to close any gaps (red circle). Do this for all graphs generated. C. Use the magic wand tool (indicated by bold red arrow) to measure the area within the intensity plot by clicking inside the enclosed plot area (cursor indicated by slim red arrow). Do this for all graphs generated. A Results table will be generated with each row corresponding to each lane. D. Repeat the steps in panels A-C for the Cdk5 immunoblot. E. Divide the area result from each lane of Cdk5 kinase activity by the area result for the same lane of the Cdk5 immunoblot. Graph and analyze as needed.

Notes

  1. Consult your institution’s radiation safety office before beginning this protocol. They can help you set up an isolated station for handling the radioactive materials as well as teach proper technique. They also likely have required protocols for ordering and disposing of radioactive materials.
  2. The detergent type or concentration in the lysis buffer may have to be modified to efficiently lyse your cells of interest. Time of lysis may also be an important factor. All subsequent buffers should contain 0.1% NP-40 to preserve the Cdk5/activator complexes.
  3. We include two obligate controls in this protocol: 1) active Cdk5/p25 complex as a positive control; 2) kinase buffer only group as a negative control to ensure reagent purity. Other controls which are specific to your experiment should be considered.
  4. This assay should be used in combination with Western blot for phosphorylated and total levels of known Cdk5 targets in lysates of your cells to confirm any changes observed in Cdk5 activity.
  5. When rescuing expression of a target of siRNA-mediated knockdown (for example, p39 in this protocol), it is optimal to use an expression vector (p39F) that contains silent point mutations in the sequence which is targeted by the siRNA (p39si). This will make the rescue expression vector resistant to the siRNA.

Recipes

  1. 1x transfer buffer (1,000 ml)
    1. Weigh 3.03 g of Tris-HCl
    2. Add 14.4 g of glycine
    3. Add 700 ml of sterile water
    4. Add 200 ml of methanol
    5. Add sterile water to a final volume of 1,000 ml
  2. 10x phosphate-buffered saline (PBS) (1,000 ml)
    1. Weigh 14.4 g of Na2HPO4, 2.4 g KH2PO4, 2.0 g KCl, and 80.0 g NaCl
    2. Add 800 ml of sterile water
    3. Stir until dissolved
    4. Bring the final volume to 1,000 ml using sterile water
  3. 1x phosphate-buffered saline (PBS) (500 ml)
    1. Add 50 ml of 10x PBS
    2. Bring final volume to 500 ml using sterile water
  4. 2 M Tris-HCl (pH 7.5) (500 ml)
    1. Weigh 121.14 g of Tris-base powder in a 500 ml glass bottle
    2. Add 450 ml of sterile water
    3. Stir until dissolved
    4. Adjust the pH to 7.5 using concentrated HCl
    5. Bring the final volume to 500 ml using sterile water
  5. 50 mM Tris-HCl (pH 7.5) (50 ml)
    1. Add 1.25 ml of 2 M Tris-HCl (pH 7.5)
    2. Bring final volume to 50 ml using sterile water
  6. 5 M NaCl (500 ml)
    1. Weigh 146.1 g of NaCl in a 500 ml glass bottle
    2. Add 450 ml of sterile water
    3. Stir until dissolved
    4. Bring the final volume to 500 ml using sterile water
  7. 0.5 M EDTA (pH 8.0) (50 ml)
    1. Weigh 7.31 g of EDTA powder in a 50 ml conical tube
    2. Add 450 ml of sterile water
    3. Stir and heat until dissolved
    4. Adjust the pH to 8.0 using NaOH tablets to aid dissolution
    5. Bring the final volume to 50 ml using sterile water
  8. 0.5 M EGTA (pH 8.0) (50 ml)
    1. Weigh 9.51 g of EGTA powder in a 50 ml conical tube
    2. Add 450 ml of sterile water
    3. Stir and heat until dissolved
    4. Adjust the pH to 8.0 using NaOH tablets to aid dissolution
    5. Bring the final volume to 50 ml using sterile water
  9. 1 M MOPS (pH 7.0) (500 ml)
    1. Weigh 104.63 g of MOPS in a 500 ml glass bottle
    2. Add 450 ml of sterile water
    3. Stir until dissolved
    4. Adjust pH to 7.0 using NaOH tablets
    5. Bring the final volume to 500 ml using sterile water
  10. 1 M MgCl2 (10 ml)
    1. Weigh 0.95 g of MgCl2 in a 15 ml conical tube
    2. Add 9.5 ml of sterile water
    3. Shake until dissolved
    4. Bring the final volume to 10 ml using sterile water
  11. 1 M NaF (10 ml)
    1. Weigh 0.42 g of NaF in a 15 ml conical tube
    2. Add 9.5 ml of sterile water
    3. Shake until dissolved
    4. Bring the final volume to 10 ml using sterile water
  12. 20% NP-40
    1. Mix 2 ml NP-40 with 8 ml sterile water
    2. Vortex and heat at 30 °C until well mixed
  13. 100 mM PMSF (1 ml)
    1. Weigh 0.017 g of PMSF in a 1.5 ml tube
    2. Add 1 ml 100% ethanol
    3. Mix until dissolved
  14. 1 mg/ml pepstatin A (1 ml)
    1. Weigh 1 mg of pepstatin A in a 1.5 ml tube
    2. Add 1 ml methanol
    3. Mix until dissolved
  15. 1 mg/ml leupeptin (1 ml)
    1. Weigh 1 mg of leupeptin in a 1.5 ml tube
    2. Add 1 ml sterile water
    3. Mix until dissolved
  16. 1 mg/ml aprotinin (1 ml)
    1. Weigh 1 mg of aprotinin in a 1.5 ml tube
    2. Add 1 ml sterile water
    3. Mix until dissolved
  17. 50% slurry of Protein A Sepharose CL-4B
    1. Decant ethanol from beads
    2. Resuspend beads in 50 mM Tris-HCl to generate a 75% bead slurry
    3. Centrifuge beads at 1,000 x g for 2 min
    4. Decant supernatant from beads
    5. Repeat steps 17b-17d
    6. Resuspend beads in 50 mM Tris-HCl to generate a 50% bead slurry
  18. Cdk5 lysis buffer (stock) (50 ml)
    1.25 ml of 2 M Tris-HCl (pH 7.5) to a 50 ml conical tube
    2.5 ml of 5 M NaCl
    100 µl of 0.5 M EDTA (pH 8.0)
    Bring final volume to 50 ml using sterile water
  19. Cdk5 lysis buffer (working) (5 ml)
    5 ml of Cdk5 lysis buffer (stock) to a 50 ml conical tube
    250 µl of 100 mM Na3VO4
    25 µl of 1 M NaF
    25 µl of 20% NP-40
    50 µl of 100 mM PMSF
    5 µl of pepstatin
    5 µl of leupeptin
    5 µl of aprotinin
  20. Cdk5 kinase buffer (stock) (50 ml)
    1 ml of 1 M MOPS (pH 7.0) to a 50 ml conical tube
    250 µl of 1 M MgCl2
    10 µl of 0.5 M EDTA (pH 8.0)
    10 µl of 0.5 M EGTA (pH 8.0)
    Bring final volume to 50 ml using sterile water
  21. Cdk5 kinase buffer (washes) (5 ml)
    5 ml of Cdk5 kinase buffer (stock) to a 15 ml conical tube
    50 µl of PMSF
  22. Cdk5 kinase buffer (assay) (1 ml)
    1 ml of Cdk5 kinase buffer (stock) to a 15 ml conical tube
    50 µl of PMSF
    2.5 µl of 10 mM ATP
    5 µl of 20 µg/µl histone H1
  23. MOPS dilution buffer (50 ml)
    1 µl of 1 M MOPS buffer to a 50 ml conical tube
    100 µl of 0.5 M EDTA (pH 8.0)
    50 µl of 10% Brij-35 solution
    2.5 ml of 100% glycerol
    50 µl of 2-mercaptoethanol
    50 mg BSA
    Bring final volume to 50 ml using sterile water
  24. 5x reaction buffer (50 ml)
    2 µl of 1 M MOPS buffer to a 50 ml conical tube
    100 µl of 0.5 M EDTA (pH 8.0)
  25. 50 mM magnesium acetate buffer (MgOAc) (15 ml)
    Weigh 0.11 g in a 15 ml conical tube
    Add sterile water to bring the final volume to 15 ml
  26. 1x phosphate-buffered saline/0.1% Tween-20 (PBS-T) (500 ml)
    50 ml of 10x PBS
    1 ml of 100% Tween-20
    Bring final volume to 500 ml using sterile water
  27. 5x Laemmli buffer
    1 ml glycerol
    1 g SDS
    1.56 ml 2M Tris-HCl
    2.5 ml β-mercaptoethanol
    1 ml 0.2% bromophenol blue
  28. 0.2% bromophenol blue
    0.02 g bromophenol blue in a 15 ml conical tube
    10 ml sterile water

Acknowledgments

This work was supported by NIH/NINDS R01NS093016 and R01NS056097, NMSSRG 4010-A, and an Emory University Research Committee grant (YF). ANB was supported by NIH training grant T32GM008602. This protocol was adapted based on previous studies (Humbert et al., 2000; Yamada et al., 2007; Bankston et al., 2013).

References

  1. Bankston, A. N., Li, W., Zhang, H., Ku, L., Liu, G., Papa, F., Zhao, L., Bibb, J. A., Cambi, F., Tiwari-Woodruff, S. K. and Feng, Y. (2013). p39, the primary activator for cyclin-dependent kinase 5 (Cdk5) in oligodendroglia, is essential for oligodendroglia differentiation and myelin repair. J Biol Chem 288(25): 18047-18057.
  2. Asada, A., Yamamoto, N., Gohda, M., Saito, T., Hayashi, N. and Hisanaga, S. (2008). Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cyclin-dependent kinase 5 complexes. J Neurochem 106(3): 1325-1336.
  3. He, X., Takahashi, S., Suzuki, H., Hashikawa, T., Kulkarni, A. B., Mikoshiba, K. and Ohshima, T. (2011). Hypomyelination phenotype caused by impaired differentiation of oligodendrocytes in Emx1-cre mediated Cdk5 conditional knockout mice. Neurochem Res 36(7): 1293-1303.
  4. Hisanaga, S. and Saito, T. (2003). The regulation of cyclin-dependent kinase 5 activity through the metabolism of p35 or p39 Cdk5 activator. Neurosignals 12(4-5): 221-229.
  5. Humbert, S., Dhavan, R. and Tsai, L. (2000). p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton. J Cell Sci 113 (Pt 6): 975-983.
  6. Ko, J., Humbert, S., Bronson, R. T., Takahashi, S., Kulkarni, A. B., Li, E. and Tsai, L. H. (2001). p35 and p39 are essential for cyclin-dependent kinase 5 function during neurodevelopment. J Neurosci 21(17): 6758-6771.
  7. Miyamoto, Y., Yamauchi, J., Chan, J. R., Okada, A., Tomooka, Y., Hisanaga, S. and Tanoue, A. (2007). Cdk5 regulates differentiation of oligodendrocyte precursor cells through the direct phosphorylation of paxillin. J Cell Sci 120(Pt 24): 4355-4366.
  8. Miyamoto, Y., Yamauchi, J. and Tanoue, A. (2008). Cdk5 phosphorylation of WAVE2 regulates oligodendrocyte precursor cell migration through nonreceptor tyrosine kinase Fyn. J Neurosci 28(33): 8326-8337.
  9. Ohshima, T., Ward, J. M., Huh, C. G., Longenecker, G., Veeranna, Pant, H. C., Brady, R. O., Martin, L. J. and Kulkarni, A. B. (1996). Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc Natl Acad Sci U S A 93(20): 11173-11178.
  10. Sato, K., Zhu, Y. S., Saito, T., Yotsumoto, K., Asada, A., Hasegawa, M. and Hisanaga, S. (2007). Regulation of membrane association and kinase activity of Cdk5-p35 by phosphorylation of p35. J Neurosci Res 85(14): 3071-3078.
  11. Tang, X. M., Strocchi, P. and Cambi, F. (1998). Changes in the activity of cdk2 and cdk5 accompany differentiation of rat primary oligodendrocytes. J Cell Biochem 68(1): 128-137.
  12. Tsai, L. H., Delalle, I., Caviness, V. S. Jr., Chae, T. and Harlow, E. (1994). p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371(6496): 419-23.
  13. Yamada, M., Saito, T., Sato, Y., Kawai, Y., Sekigawa, A., Hamazumi, Y., Asada, A., Wada, M., Dio, H. and Hisanaga, S. (2007). Cdk5-p39 is a labile complex with the similar substrate specificity to Cdk5-p35. J Neurochem 102: 1477-87
  14. Yang, Y., Wang, H., Zhang, J., Luo, F., Herrup, K., Bibb, J. A., Lu, R. and Miller, R. H. (2013). Cyclin dependent kinase 5 is required for the normal development of oligodendrocytes and myelin formation. Dev Biol 378(2): 94-106.
  15. Zheng, M., Leung, C. L. and Liem, R. K. (1998). Region-specific expression of cyclin-dependent kinase 5 (cdk5) and its activators, p35 and p39, in the developing and adult rat central nervous system. J Neurobiol 35(2): 141-159.

简介

Cdk5活性受两种激活蛋白p35和p39(Tsai et al。,1994; Zheng et al。,1998; Humbert等人)的量的调节,2000)。 p35-Cdk5和p39-Cdk5复合物对盐和洗涤剂浓度的敏感性不同(Hisanaga和Saito,2003; Sato et al。,2007; Yamada等人, 2007; Asada 等人,2008)。 Cdk5激活可以通过Cdk5与其结合的激活剂的免疫沉淀直接测量,随后进行Cdk5激酶测定。在该方案中,用于细胞裂解和免疫沉淀的缓冲液旨在保持p35-和p39-Cdk5复合物以评估总Cdk5活性。裂解细胞,并在核后上清液中测定蛋白浓度。 Cdk5在实验组之间从等量的总蛋白免疫沉淀。然后进行洗涤以除去外来蛋白质并平衡激酶缓冲液中的Cdk5-活化剂复合物。然后将Cdk5与组蛋白H1孵育,组蛋白H1是Cdk5和[γ- 32 P] ATP在体外成功建立的靶标。反应通过SDS-PAGE解析并转移到膜上,用于可视化H1磷酸化和免疫沉淀的Cdk5水平的免疫印迹。我们已经使用该测定来建立p39作为少突神经胶质谱系中Cdk5的主要活化剂。然而,该测定法适用于对裂解条件进行适当调整的其它细胞谱系或组织。
【背景】虽然Cdk5通常与神经元功能相关,但最近的工作已经证明Cdk5也可以调节少突胶质细胞祖细胞(OPC)的发育(Tang等人,1998; Miyamoto等人 ,2007年和2008年)。 Cdk5功能对于OPC迁移和分化是至关重要的,Cdk5的丧失导致CNS脊髓灰质沉积(Miyamoto et al。,2007和2008; He et al。,2010; Yang 等人,2013)。然而,调节神经元和OLs中Cdk5功能的分子机制仍然难以捉摸。 Cdk5的活性由两个激活物同源物p35和p39(Tsai等人,1994; Zheng等人,1998; Humbert > et al。,2000)。在缺乏p35和p39的小鼠中观察到的胚胎脑发育和围产期致死性中的缺陷几乎与Cdk5无效小鼠中的缺陷相同(Ohshima等人,1996; Ko等人) ,2001),表明p35和p39是脑中Cdk5的唯一活化剂。我们发现,与p35在神经元中激活Cdk5的主要作用相反,p39是少突胶质细胞(OL)中的主要Cdk5激活剂,其中p35表达可忽略。使用这种活性Cdk5免疫沉淀和激酶测定,我们证明Cdk5活性在具有siRNA介导的p39敲低的OL中几乎完全消融。以前的工作确定了p35和p39对高洗涤剂和盐浓度的不同灵敏度(Hisanaga和Saito,2003; Sato et al。,2007; Yamada等人,2007 ; Asada 等人,2008)。基于这些报告,该方案被开发用于尝试并保存p35-和p39-Cdk5复合物以测量总Cdk5活性,而不管活化剂如何。我们的工作进一步表明,p39对于OL分化和髓鞘修复至关重要,p35的上调掩盖了髓鞘发育过程中p39功能的损失。测量细胞Cdk5活性,结合Cdk5靶磷酸化的免疫印迹,提供了一种识别Cdk5激活新方案的工具。

关键字:激酶活性测定, Cdk5, 蛋白质印迹, 免疫印迹, 辐射

材料和试剂

  1. 电池升降机(Corning,目录号:3008)
  2. Corning无菌60毫米细胞培养皿(康宁,目录号:3261)
  3. Corning无菌100毫米细胞培养皿(康宁,目录号:3262)
  4. 15ml锥形管(Denville Scientific,目录号:C1018-P)
  5. 微型载玻片(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4951PLUS4)
  6. 微盖玻璃(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:25X54I24901)
  7. 1.7ml管(Denville Scientific,SlipTech TM,目录号:C19033)
  8. 50ml锥形管(Denville Scientific,目录号:1005513)
  9. 硝酸纤维素或PVDF膜(GE Healthcare,目录号:RPN82D或10600021)
  10. 高压灭菌微量吸头(Denville Scientific,Woodpecker Reloads TM ,目录号:P2102-NB,P2101-N,P2109)
  11. X射线胶片(Denville Scientific,Hyblot ES ®,目录号:E3218)
  12. 台盼蓝(Sigma-Aldrich,目录号:302643)
  13. BCA试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23235)
  14. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  15. 抗Cdk5抗体(Santa Cruz C-8)(Santa Cruz Biotechnology,目录号:sc-173)
  16. 抗Cdk5抗体(Cell Signaling Technology,目录号:2506)
  17. 兔IgG抗体(Vector Laboratories,目录号:I-1000)
  18. 山羊抗兔 - 辣根过氧化物酶(HRP)(Jackson ImmunoResearch,目录号:111-035-003)
  19. 10mM ATP(Thermo Fisher Scientific,目录号:PV3227)
  20. Cdk5 / p25,活性复合物(EMD Millipore,目录号:14-516)
  21. [γ- 32 P] ATP(PerkinElmer,目录号:BLU002Z250UC)
  22. 组蛋白H1(EMD Millipore,目录号:14-155)
  23. 12%聚丙烯酰胺凝胶(Bio-Rad Laboratories,目录号:4561043)
  24. 增强的化学发光试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:32106)
  25. Tris / Glycine / SDS缓冲液(Bio-Rad Laboratories,目录号:1610732)
  26. 甲醇(Sigma-Aldrich,目录号:34860)
  27. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  28. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  29. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  30. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  31. Tris碱(Sigma-Aldrich,目录号:RDD008)
  32. 浓HCl(Sigma-Aldrich,目录号:295426)
  33. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS)
  34. 氢氧化钠(NaOH)片剂(Sigma-Aldrich,目录号:S8045)
  35. 乙二醇 - 双(β-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  36. 3-(N-吗啉代)丙磺酸(MOPS)(Sigma-Aldrich,目录号:RDD003)
  37. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  38. 氟化钠(NaF)(Sigma-Aldrich,目录号:S7920)
  39. NP-40 / IGEPAL ® CA-630(Sigma-Aldrich,目录号:I3021)
  40. 聚甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  41. 100%乙醇(Sigma-Aldrich,目录号:E7023)
  42. Pepstatin(Sigma-Aldrich,目录号:P4265)
  43. 亮肽素(Sigma-Aldrich,目录号:L2884)
  44. 抑肽酶(Sigma-Aldrich,目录号:A1153)
  45. 蛋白A Sepharose CL-4B(GE Healthcare,目录号:17-0780-01)
  46. 预活化的原钒酸钠(100mM Na 3 VO 4)(New England Biolabs,目录号:P0758L)
  47. 10%Brij TM -35(Thermo Fisher Scientific,Thermo Scientific TM,目录号:28316)
  48. 甘油(Sigma-Aldrich,目录号:G5516)
  49. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  50. 乙酸镁(MgOAc)(Sigma-Aldrich,目录号:M5661)
  51. 吐温-20(Sigma-Aldrich,目录号:P1379)
  52. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126)
  53. 1x传输缓冲区(见配方)
  54. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  55. 1x磷酸缓冲盐水(PBS)(见食谱)
  56. 2 M Tris-HCl(pH 7.5)(参见食谱)
  57. 50mM Tris-HCl(pH7.5)(参见食谱)
  58. 5 M NaCl(见食谱)
  59. 0.5 M EDTA(pH 8.0)(参见食谱)
  60. 0.5 M EGTA(pH 8.0)(参见食谱)
  61. 1 M MOPS(pH 7.0)(见配方)
  62. 1 M MgCl 2(见配方)
  63. 1 M NaF(见配方)
  64. 20%NP-40(见配方)
  65. 100 mM PMSF(见配方)
  66. 1 mg / ml胃蛋白酶抑素A(见食谱)
  67. 1 mg / ml亮抑酶肽(见配方)
  68. 1 mg / ml抑肽酶(见食谱)
  69. 蛋白A Sepharose CL-4B的50%浆液(参见食谱)
  70. Cdk5裂解缓冲液(库存)(见配方)
  71. Cdk5裂解缓冲液(工作)(见配方)
  72. Cdk5激酶缓冲液(库存)(见配方)
  73. Cdk5激酶缓冲液(洗涤)(参见食谱)
  74. Cdk5激酶缓冲液(测定)(见配方)
  75. MOPS稀释缓冲液(见配方)
  76. 5x反应缓冲液(见配方)
  77. 50mM醋酸镁缓冲液(MgOAc)(参见食谱)
  78. 1x磷酸缓冲盐水/ 0.1%Tween-20(PBS-T)(参见食谱)
  79. 5x Laemmli缓冲液(参见食谱)
  80. 0.2%溴酚蓝(见食谱)

设备

  1. 血细胞计数仪(Hausser Scientific,目录号:3100)
  2. PIPETMAN Classic TM 移液器(Gilson,型号:P10,目录号:F144802)
  3. PIPETMAN Classic TM 移液器(Gilson,型号:P20,目录号:F123600)
  4. PIPETMAN Classic TM 移液器(Gilson,型号:P200,目录号:F123601)
  5. PIPETMAN Classic TM 移液器(Gilson,型号:P1000,目录号:F123602)
  6. 冷冻台式离心机,用于15 ml锥形管(Jouan,型号:CT422)
  7. 在4℃的冷室中的1.5ml管的桌面离心机(Eppendorf,型号:5415D)
  8. 倒置光学显微镜(Olympus,型号:CK30)
  9. 认证盖革计数器(W.B.Johnson Instruments,型号:GSM-110)
  10. 有机玻璃屏蔽(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:6700-1812)
  11. 磷光成像盒(Thomas Scientific,目录号:C993J84)
    制造:bioWORLD,目录号:43121008-1。
  12. 放射自显影盒(Denville Scientific,目录号:E3122)
  13. 500毫升玻璃瓶(康宁,PYREX ®,目录号:1397-500)

软件

  1. GraphPad Prism(GraphPad软件)
  2. ImageJ( https://imagej.nih.gov/ij

程序

注意:

  1. 步骤A1-A4应尽可能快地进行,以避免在洗涤期间细胞蛋白质的损失。
  2. 该方案旨在保护Cdk5 /激活剂复合物,同时仍然保持足够的严格性以合理地裂解细胞并从免疫沉淀的复合物中除去非特异性蛋白质。
  3. 一旦在步骤D3b中添加了[γ- 32 P] ATP,则在本协议的其余部分,必须根据您所在机构的辐射安全协议来处理所有丢弃的解决方案。 >
  1. 产生细胞裂解物
    1. 细胞培养物应与适当的治疗组和对照组一起繁殖。
    2. 从平板吸出培养基,并用PBS轻轻冲洗细胞培养物(参见食谱)两次。从最后一次洗涤中取出PBS。
    3. 使用细胞提升器从培养板中分离细胞。通过用适当体积的PBS冲洗该平板来收集分离的细胞(1毫升为60毫米的平板,3毫升为100毫米的板,)。
    4. 将细胞悬浮液转移到15 ml锥形管中
    5. 在4℃下以3,500×g离心细胞悬浮液5分钟。
    6. 如果需要,重复步骤A3-A5。
    7. 将细胞沉淀物以15%w / v重悬于裂解缓冲液(参见食谱)。
    8. 在冰上孵育裂解物10分钟,同时定期搅动
    9. 通过将10μl裂解物1:1样品与台盼蓝混合来确认裂解。将10μl该混合物移至显微镜载玻片上,并将盖玻片置于10μl液滴上。良好的裂解将出现与细胞内不再包含的完整核一样多的细胞核。未分配的单元格将显示为具有蓝色轮廓的球体。如果细胞核裂解,将会存在许多棕色纤维,这可能会对您的免疫沉淀的纯度产生负面影响。未分解细胞的示例图像和良好的裂解在图1中给出。
    10. 将10%的裂解物保留为“输入”,加入5x Laemmli缓冲液(参见食谱)至输入至最终浓度为1x,并将样品储存在冰上直到步骤D9。


      图1.裂解确认。左侧,用台盼蓝染色的未分解细胞的示例图像。注意每个单元格周围的蓝色轮廓和轻微晕圈。对,来自裂解细胞的完整核的示例图像
  2. 准备样品进行免疫沉淀
    1. 在4℃下以10,000×g离心裂解物10分钟
    2. 将上清液转移至新鲜管中
    3. 加入50μl蛋白A珠的50%浆液(或用于免疫沉淀的任何种类的珠子)至裂解液。
    4. 在4℃下旋转或摇动珠粒和裂解物的混合物30分钟,以预先清除非特异性结合珠粒的蛋白质裂解物。
    5. 在4℃下将混合物以3,000xg离心2分钟。
    6. 将上清液转移至新鲜管中
    7. 使用标准蛋白质定量方法(例如Bradford或BCA试剂盒)量化裂解物中蛋白质的浓度,
      1. 通过使用无菌水稀释,产生1mg / ml,0.5mg / ml,0.25mg / ml,0.125mg / ml和0mg / ml牛血清白蛋白(BSA)的标准曲线。
      2. 稀释裂解液(细胞裂解液1:2至1:10),使样品蛋白浓度落在BSA标准曲线范围内。
      3. 按照制造商的说明记录裂解液中蛋白质的浓度。注意R 2 值,因为它表示蛋白质浓度定量的准确性(0.9以上是期望的)。
      4. 取三个读数的平均值。

  3. 免疫沉淀Cdk5 /激活剂复合物
    1. 准备两个新鲜的1.5 ml管,从裂解物中加入1 mg总蛋白 注意:如果您的一个样品含有不足的蛋白质浓度,请将所有样品中的蛋白质总量相等,以匹配蛋白质浓度最低的样品中的蛋白质量。
    2. 加入裂解缓冲液使每个管中的总体积达到500μl
    3. 向每个样品准备的1.5 ml管中加入10μl(2μg)Santa Cruz抗Cdk5抗体(C-8)。
    4. 向每个样品准备的其他1.5 ml管中加入2μgIgG抗体。
    5. 在4℃下旋转或摇动珠粒和裂解物的混合物3小时
    6. 向每个管中加入50μl蛋白A珠的50%浆液。
    7. 在4℃下旋转或摇动珠粒和裂解物的混合物2小时
    8. 在4℃下以3000×g离心管2分钟。
    9. 取出并丢弃上清液。
    10. 加入500μl含有0.1%NP-40的裂解缓冲液
    11. 在4℃下旋转或摇动珠粒和裂解物的混合物10分钟
    12. 重复步骤C8-C11两次。
    13. 在4℃下以3000×g离心管2分钟。
    14. 取出并丢弃上清液。
    15. 加入500μl无ATP或组蛋白H1的激酶缓冲液(见配方)。
    16. 在4℃下旋转或摇动珠粒和裂解物的混合物10分钟
    17. 再次重复步骤C13-C16(图2中免疫沉淀的一般方案)。


      图2.活性Cdk5复合物的免疫沉淀方案

  4. Cdk5激酶测定
    1. 将珠重悬于25μl激酶缓冲液(含100 ng /μlH1和25μMATP)
    2. 准备与重组Cdk5 / p25阳性对照(Cdk5激酶测定的一般方案和图3中的对照)的反应。
      1. 加入14.6μlMOPS稀释缓冲液(见食谱)至1μlCdk5 / p25活化复合物(Upstate / Millipore)。
      2. 加入5μl5x反应缓冲液(参见食谱)至新鲜的1.5 ml管
      3. 加入2.5μl组蛋白H1(最终100 ng /μl)
      4. 将2.5μl稀释的Cdk5从步骤D2a添加到1.5 ml管中。
      5. 加入5μl无菌水

        图3. Cdk5激酶测定,SDS-PAGE和检测方案

    3. 准备[γ- 32 P] ATP的工作解决方案
      1. 准备47μl25 mM醋酸镁(见食谱)。
      2. 加入[γ- 32 P] ATP(终浓度0.1μCi/μl)
      3. 加入10 mM ATP,使总体积达到50μl
    4. 加入10μl步骤D3中的 32 P混合物至步骤D2中制备的阳性对照反应。
    5. 从步骤D3向步骤D1的每个IP反应添加2μl的 32 P混合物
    6. 将25μl激酶缓冲液与来自步骤D3的2μl 32 P混合物作为阴性对照。
    7. 将所有反应在30°C下孵育30分钟
    8. 在每个反应中加入5μl5x Laemmli缓冲液。
    9. 可选:在-20°C存储反应。
    10. 立即用12.5%SDS-PAGE解决。
    11. 不要让染料前面离开凝胶底部。这将减少运行缓冲器或设备的放射性污染。
    12. 解决反应后,在底部切断染料(含有自由基)和堆积凝胶。
    13. 将分解的蛋白转移到硝酸纤维素或PVDF膜上
    14. 在-80℃下将膜暴露于膜或荧光屏上(参见图4中的实施例)。


      图4.免疫沉淀中Cdk5激酶活性和蛋白水平的检测从空载体(Ctrlsi + pc),空载体p39 siRNA转染的细胞(Ctrlsi + pc)转染的细胞进行的Cdk5免疫沉淀和激酶测定的结果p39si + pc),具有p39-FLAG(p39si + p39F)的p39 siRNA。仅包含激酶缓冲液作为阴性对照。在该图中,外源表达的p39-FLAG逆转了siRNA介导的内源性p39敲低引起的对Cdk5活性的影响。这项研究最初发表在“生物化学杂志”上。 Bankston AN,Li W,Zhang H,Ku L,Liu G,Papa F,Zhao L,Bibb JA,Cambi F,Tiwari-Woodruff SK,Feng Y.P39,a primary activator for cyclin-dependent kinase 5(Cdk5)in少突胶质细胞是少突胶质细胞分化和髓鞘修复所必需的。 J Biol Chem。 2013; 288(25):18047-57。 &复制;美国生物化学与分子生物学学会。

  5. 进行蛋白质印迹以评估免疫沉淀
    1. 用PBS-T中10%的无脂牛奶将膜孵育1小时(参见食谱)
    2. 丢弃10%牛奶溶液。
    3. 在含有1:1,000稀释的抗Cdk5抗体(Cell Signaling Technology)的PBS-T中的2%无脂奶上孵育4℃过夜。
    4. 弃去含有抗Cdk5抗体的2%乳液。
    5. 通过在PBS-T中孵育10分钟来洗涤膜。
    6. 弃去PBS-T洗液。
    7. 再次重复步骤E5-E6。
    8. 在含有1:5000稀释的HRP缀合的抗兔IgG抗体(Jackson ImmunoResearch)的PBS-T中的2%非脂肪乳中孵育该膜1小时。
    9. 通过在PBS-T中孵育10分钟来洗涤膜。
    10. 弃去PBS-T洗液。
    11. 根据制造商的方案,用增强型化学发光溶液孵育膜。
    12. 将膜暴露在薄膜上(见图4中的例子)

数据分析

  1. 对于实验设计,对于给定的实验,免疫沉淀和激酶测定至少进行三次。
  2. 使用ImageJ进行光密度分析。对于每个样品,步骤D13中测量的组蛋白H1磷酸化的强度被归一化为在步骤E12中测量的Cdk5蛋白的强度(参见图5)。
  3. 对于统计分析,我们使用GraphPad Prism软件。为了比较两个样本,使用了一个双尾Student's 测试。为了比较三个或更多个样本,使用单因素方差分析与事后Tukey校正。


    图5. ImageJ中Cdk5激酶活性和蛋白质水平的测量和归一化。 A.在Cdk5激酶活性的第一泳道周围绘制一个盒子(黄色固体盒)。单击分析凝胶菜单下的选择第一车道。将盒子移动到Cdk5激酶活性的第二道上(黄色虚线框)。单击分析凝胶菜单下的选择第二通道。将盒子移动到Cdk5激酶活性的每个剩余通道上,单击每个泳道下的“分析凝胶”菜单下的“选择第二通道”。单击“分析凝胶”菜单下的“绘图”。 B.将为每个车道生成图。使用线条工具(用粗体红色箭头表示)在图形的底部绘制一条线(由细长的红色箭头表示的光标绘制线)。确保关闭任何间隙(红色圆圈)。对所生成的所有图形执行此操作。 C.使用魔术棒工具(用粗体红色箭头表示)通过点击封闭绘图区域内的光线(由细长的红色箭头指示的光标)来测量强度图中的区域。对所生成的所有图形执行此操作。将生成与每个行对应的每行的“结果”表。 D.重复图A-C中Cdk5免疫印迹的步骤。 E.将Cdk5激酶活性的每个泳道的区域结果除以Cdk5免疫印迹的同一泳道的面积结果。根据需要进行图形化和分析

笔记

  1. 在开始本协议之前,请咨询贵机构的辐射安全办公室。他们可以帮助您建立一个处理放射性物质的隔离站,并教授适当的技术。他们也可能需要订购和处置放射性物质的协议。
  2. 溶解缓冲液中的洗涤剂类型或浓度可能需要修改才能有效地溶解您感兴趣的细胞。裂解时间也可能是一个重要因素。所有随后的缓冲液应含有0.1%的NP-40以保护Cdk5 /活化剂复合物
  3. 我们在本协议中包括两项专项控制:1)活性Cdk5 / p25复合物作为阳性对照; 2)激酶缓冲液仅作为阴性对照组,以确保试剂纯度。应考虑与实验相关的其他控制。
  4. 该测定应与Western印迹结合使用,以确定细胞裂解液中已知Cdk5靶标的磷酸化和总水平,以确认Cdk5活性中观察到的任何变化。
  5. 当拯救siRNA介导的敲低靶标(例如本方案中的p39)的表达时,使用在siRNA(p39si)靶向的序列中含有沉默点突变的表达载体(p39F)是最佳的。这将使救援表达载体对siRNA有抗性。

食谱

  1. 1x转移缓冲液(1,000ml)
    1. 称量3.03克Tris-HCl
    2. 加入14.4 g甘氨酸
    3. 加入700 ml无菌水
    4. 加入甲醇200毫升
    5. 加入无菌水至最终体积为1000 ml
  2. 10倍磷酸缓冲盐水(PBS)(1000毫升)
    1. 称取14.4g的Na 2 HPO 4,2.4g KH 2 PO 4,2.0g KCl和80.0 g NaCl
    2. 加入800 ml无菌水
    3. 搅拌直至溶解
    4. 使用无菌水使最终体积达到1000毫升
  3. 1x磷酸缓冲盐水(PBS)(500ml)
    1. 加入50毫升10倍的PBS
    2. 使用无菌水将终体积至500毫升
  4. 2M Tris-HCl(pH7.5)(500ml)
    1. 称取121.14克Tris-base粉末在500毫升玻璃瓶中
    2. 加入无菌水450毫升
    3. 搅拌直至溶解
    4. 使用浓HCl将pH调节至7.5
    5. 使用无菌水使最终体积达到500 ml
  5. 50mM Tris-HCl(pH7.5)(50ml)
    1. 加入1.25ml 2M Tris-HCl(pH7.5)
    2. 使用无菌水使最终体积达到50ml
  6. 5M NaCl(500ml)
    1. 称量146.1克NaCl在500毫升玻璃瓶
    2. 加入无菌水450毫升
    3. 搅拌直至溶解
    4. 使用无菌水使最终体积达到500 ml
  7. 0.5M EDTA(pH8.0)(50ml)
    1. 称取7.31g EDTA粉末在50ml锥形管中
    2. 加入无菌水450毫升
    3. 搅拌和加热直至溶解
    4. 使用NaOH片调节pH至8.0以帮助溶解
    5. 使用无菌水使最终体积达到50ml
  8. 0.5M EGTA(pH8.0)(50ml)
    1. 称取9.51克EGTA粉末在50ml锥形管中
    2. 加入无菌水450毫升
    3. 搅拌和加热直至溶解
    4. 使用NaOH片调节pH至8.0以帮助溶解
    5. 使用无菌水使最终体积达到50ml
  9. 1M MOPS(pH7.0)(500ml)
    1. 称量104.63克MOPS在一个500毫升玻璃瓶
    2. 加入无菌水450毫升
    3. 搅拌直至溶解
    4. 使用NaOH片将pH调至7.0
    5. 使用无菌水使最终体积达到500 ml
  10. 1M MgCl 2(10ml)
    1. 在15毫升锥形管中称量0.95克MgCl 2 2
    2. 加入9.5 ml无菌水
    3. 摇动直到溶解
    4. 使用无菌水使最终体积达到10ml
  11. 1M NaF(10ml)
    1. 称取0.42 g NaF在15 ml锥形管中
    2. 加入9.5 ml无菌水
    3. 摇动直到溶解
    4. 使用无菌水使最终体积达到10ml
  12. 20%NP-40
    1. 混合2毫升NP-40与8毫升无菌水
    2. 涡旋并在30°C下加热,直至混合好
  13. 100mM PMSF(1ml)
    1. 称量0.017 g PMSF在1.5 ml管中
    2. 加入1 ml 100%乙醇
    3. 混合直到溶解
  14. 1mg / ml胃蛋白酶抑素A(1ml)
    1. 称量1毫克胃蛋白酶抑制剂A在1.5毫升管中
    2. 加入1 ml甲醇
    3. 混合直到溶解
  15. 1毫克/毫升亮抑素(1毫升)
    1. 称量1毫克亮抑素在1.5毫升的管中
    2. 加入1 ml无菌水
    3. 混合直到溶解
  16. 1mg / ml抑肽酶(1ml)
    1. 称取1毫克的抑肽酶在1.5毫升的管中
    2. 加入1 ml无菌水
    3. 混合直到溶解
  17. 蛋白A Sepharose CL-4B的50%浆液
    1. 来自珠子的滗析乙醇
    2. 将珠重悬于50mM Tris-HCl中以产生75%的珠浆料
    3. 离心珠以1,000×g离心2分钟
    4. 来自珠粒的上清液
    5. 重复步骤17b-17d
    6. 将珠子重悬于50mM Tris-HCl中以产生50%珠粒浆料
  18. Cdk5裂解缓冲液(原液)(50ml)
    将1.25ml 2M Tris-HCl(pH7.5)加至50ml锥形管中 2.5 ml 5 M NaCl 100μl0.5M EDTA(pH8.0)
    使用无菌水使最终体积达到50ml
  19. Cdk5裂解缓冲液(工作)(5 ml)
    将5ml的Cdk5裂解缓冲液(储备)加入到50ml锥形管中 250μl100mM Na 3 VO 4< 4>
    25μl1 M NaF
    25μl20%NP-40
    50μl100 mM PMSF
    5微升胃蛋白酶抑制素
    5微升亮肽素
    5微升抑肽酶
  20. Cdk5激酶缓冲液(原液)(50ml)
    1毫升1M MOPS(pH 7.0)至50ml锥形管 250μl的1M MgCl 2
    10μl0.5M EDTA(pH8.0)
    10μl0.5M EGTA(pH8.0)
    使用无菌水使最终体积达到50ml
  21. Cdk5激酶缓冲液(洗涤)(5ml)
    将5ml的Cdk5激酶缓冲液(储备)加到15ml锥形管中 50μlPMSF
  22. Cdk5激酶缓冲液(测定)(1ml)
    将1ml Cdk5激酶缓冲液(储备)加到15ml锥形管中 50μlPMSF
    2.5μl10 mM ATP
    5μl20μg/μl组蛋白H1
  23. MOPS稀释缓冲液(50ml)
    将1μl1M MOPS缓冲液加入50ml锥形管中 100μl0.5M EDTA(pH8.0)
    50μl10%Brij-35溶液
    2.5毫升100%甘油
    50μl2-巯基乙醇
    50毫克BSA
    使用无菌水使最终体积达到50ml
  24. 5x反应缓冲液(50ml)
    将2μl1M MPS缓冲液加入50ml锥形管中 100μl0.5M EDTA(pH8.0)
  25. 50mM醋酸镁缓冲液(MgOAc)(15ml) 称取0.11 g在15 ml锥形管中 加入无菌水使最终体积达到15 ml
  26. 1x磷酸缓冲盐水/ 0.1%Tween-20(PBS-T)(500ml) 50毫升10倍PBS
    1ml 100%Tween-20
    使用无菌水将终体积至500毫升
  27. 5x Laemmli缓冲区
    1毫升甘油
    1克SDS
    1.56ml 2M Tris-HCl
    2.5mlβ-巯基乙醇
    1 ml 0.2%溴酚蓝
  28. 0.2%溴酚蓝
    0.02g溴酚蓝在15ml锥形管中 10ml无菌水

致谢

这项工作得到NIH NS053905,NMSSRG 4010-A和Emory大学研究委员会授权的支持。 AB由NIH T32GM008602支持。该协议基于以前的研究(Humbert等人,2000; Yamada等人,2007)进行了改编。

参考

  1. Asada,A.,Yamamoto,N.,Gohda,M.,Saito,T.,Hayashi,N。和Hisanaga,S。(2008)。 p39和p35的肉豆蔻酰化是活细胞周期蛋白依赖性激酶5复合物的细胞质或核定位的决定因素。 J Neurochem 106(3):1325-1336。
  2. He,X.,Takahashi,S.,Suzuki,H.,Hashikawa,T.,Kulkarni,AB,Mikoshiba,K.and Ohshima,T。(2011)。< a class =“ke-insertfile”href = “http://www.ncbi.nlm.nih.gov/pubmed/21210220”target =“_ blank”>由Emx1-cre介导的Cdk5条件敲除小鼠中少突胶质细胞分化导致的过度表型。 Neurochem Res 36(7):1293-1303。
  3. Hisanaga,S.和Saito,T。(2003)。通过p35或p39 Cdk5激活物的代谢调节细胞周期蛋白依赖性激酶5活性。神经信号 12(4-5):221-229。
  4. Humbert,S.,Dhavan,R.和Tsai,L。(2000)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/10683146”目标=“_ blank”> p39激活神经元中的cdk5,并与肌动蛋白细胞骨架相关。细胞科学 113(Pt 6):975-983。
  5. Ko,J.,Humbert,S.,Bronson,RT,Takahashi,S.,Kulkarni,AB,Li,E.和Tsai,LH(2001)。< a class =“ke-insertfile”href =“http ://www.ncbi.nlm.nih.gov/pubmed/11517264“target =”_ blank“> p35和p39在神经发育过程中对细胞周期蛋白依赖性激酶5的功能至关重要。 J Neurosci 21(17):6758-6771。
  6. Miyamoto,Y.,Yamauchi,J.,Chan,JR,Okada,A.,Tomooka,Y.,Hisanaga,S.and Tanoue,A。(2007)。< a class =“ke-insertfile”href = “http://www.ncbi.nlm.nih.gov/pubmed/18042622”target =“_ blank”> Cdk5通过paxillin的直接磷酸化来调节少突胶质细胞前体细胞的分化。 / em> 120(Pt 24):4355-4366。
  7. Miyamoto,Y.,Yamauchi,J.and Tanoue,A。(2008)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/18701695”目标=“_ blank”> WAVE2的Cdk5磷酸化调节通过非受体酪氨酸激酶Fyn的少突胶质细胞前体细胞迁移。 J Neurosci 28(33):8326-8337。
  8. Ohshima,T.,Ward,J.M.,Huh,C.G.,Longenecker,G.,Veeranna,Pant,H.C.,Brady,R.O。,Martin,L.J。和Kulkarni,A.B。(1996)。 细胞周期蛋白依赖性激酶5基因的靶向破坏导致异常皮质发生,神经元病理和围产期死亡。
    Proc Natl Acad Sci U S A 93(20):11173-11178。
  9. Sato,K.,Zhu,YS,Saito,T.,Yotsumoto,K.,Asada,A.,Hasegawa,M。和Hisanaga,S。(2007)。通过p35的磷酸化调节Cdk5-p35的膜缔合和激酶活性。 Neurosci Res 85(14):3071-3078。
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  • English
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bankston, A. N., Ku, L. and Feng, Y. (2017). Active Cdk5 Immunoprecipitation and Kinase Assay. Bio-protocol 7(13): e2363. DOI: 10.21769/BioProtoc.2363.
  2. Bankston, A. N., Li, W., Zhang, H., Ku, L., Liu, G., Papa, F., Zhao, L., Bibb, J. A., Cambi, F., Tiwari-Woodruff, S. K. and Feng, Y. (2013). p39, the primary activator for cyclin-dependent kinase 5 (Cdk5) in oligodendroglia, is essential for oligodendroglia differentiation and myelin repair. J Biol Chem 288(25): 18047-18057.
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