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Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue
从小鼠骨骼肌组织中骨骼肌细胞核的生化分离   

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Aging Cell
May 2017

Abstract

Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. The nucleus is a major site of signaling integration and gene expression regulation. However, examining nuclear processes in skeletal muscle can be difficult because myonuclei are challenging to isolate. We optimized a protocol to purify myonuclei from whole muscle tissue using ultracentrifugation over a discontinuous sucrose gradient to separate the nuclear fraction. We used these purified nuclei for downstream applications including flow cytometry and mass spectrometry. We used this method to compare the myonuclear proteome of young and old mouse hindlimb muscles (Cutler et al., 2017). This protocol may be applied to isolating myonuclei for a variety of downstream analyses such as flow cytometry, microscopy, Western blot, and proteomics.

Keywords: Fractionation (分馏), Sucrose gradient (蔗糖梯度), Magnetic isolation (磁分离), Ultracentrifugation (超速离心)

Background

Proper skeletal muscle function must be maintained for survival. One component of this maintenance is adjustments in gene expression in response to cellular needs and environmental cues. Nuclear processes modulating gene expression are a critical component in regulating cellular composition and behavior. However, myonuclear proteins involved in these processes are difficult to study because of four technical limitations. First, skeletal muscle is dense, tightly packed with contractile proteins that make up more than 60% of proteins in the tissue (Deshmukh et al., 2015; Cutler et al., 2017). These high abundance contractile proteins eclipse the far less abundant nuclear proteins. Second, the dense fibrous structure of skeletal muscle makes it difficult to dissociate without damaging nuclei, making nuclei difficult to isolate. Third, after centrifugation dense debris cosediments with nuclei, compounding the difficulty of isolating nuclei from the tissue. Fourth, skeletal muscle is comprised of multiple cell types, so nuclei isolated and nuclear proteins detected may be from myonuclei or nuclei of other cell types.

Several approaches have been optimized to enrich myonuclei from different organisms for various downstream applications. Ohkawa et al. presented a detailed protocol for isolating myonuclei from mouse tissue that was developed to maximize access of cross-linking reagent for Chromatin Immunoprecipitation (ChIP) analysis (Ohkawa et al., 2012). Wilkie and Shrimer developed a procedure to isolate the myonuclear envelope and sarcoplasmic reticulum for proteomic comparison (Wilkie and Schirmer, 2008). An approach optimized by Dimauro et al. simultaneously collected mitochondrial, nuclear, and cytoplasmic fractions to compare protein localization among different cellular compartments (Dimauro et al., 2012). While each of these approaches to enrich nuclei from skeletal muscle tissue was effective for the intended subsequent analysis, they did not prioritize isolating intact nuclei and not distinguish between myonuclei and nuclei from other cell types. An affinity-based method to selectively isolate nuclei from specific cell types was developed in Arabidopsis thaliana (Deal and Henikoff, 2011) and is now available for mice (Jankowska et al., 2016). However, this affinity-based approach requires genetic labeling of the cell types of interest, which makes it prohibitively cumbersome to examine myonuclei from multiple mouse models. We optimized an ultracentrifugation sucrose gradient-based fractionation approach that requires relatively small sample sizes, no genetic labeling, and is compatible with downstream analysis by flow cytometry and mass spectrometry. The isolated nuclei are intact, biochemically depleted of proteins from non-nuclear organelles, and 85% of nuclei are myonuclei. To isolate myonuclei more quickly we also optimized affinity-based purification using a myonuclear-specific nuclear envelope protein, Transmembrane Protein 38A (TMEM38A) (Bleunven et al., 2008; Cutler et al., 2017), and magnetic beads. This approach is more rapid and yields nuclei with comparable population purity to nuclear isolation by ultracentrifugation but with lower biochemical purity. These nuclear isolation techniques can be used to purify myonuclei from any mouse model for diverse downstream analyses.

Materials and Reagents

  1. Materials
    1. 30 ml ultraclear ultracentrifuge tubes (Beckman Coulter, catalog number: 344058 )
    2. 10 ml round bottom polypropylene tubes (Corning, Falcon®, catalog number: 352059 )
    3. 40 µm nylon mesh cell strainer (Biologix, catalog number: 15-1040 )
    4. 50 ml conical polypropylene tubes (BioExpress, catalog number: C-3394-4 )
    5. 15 cm glass Pasteur pipets (Fisher Scientific, catalog number: 13-678-20A )
    6. 20-gauge needle (Fisher Scientific, catalog number: 14-826-5C)
      Manufacturer: BD, catalog number: 305176 .
    7. 10 ml syringe (BD, catalog number: 309604 )
    8. 1.7 ml low-adhesion hydrophobic tubes (BioExpress, GeneMate, catalog number: C-3302-1 )
    9. 5 ml conical polystyrene tube with cell strainer cap (Corning, catalog number: 352235 )
    10. 0.45 µm polypropylene syringe filters (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F2500-9 )

  2. Reagents
    1. Ethanol
    2. 4,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
    3. Protein A-conjugated magnetic beads (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10001D )
    4. Anti-TMEM38A antibody (Merck, catalog number: 06-1005 )
    5. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: BP120 )
    6. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318 )
    7. Ethylene glycol-bis(β-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
    8. Sucrose (Fisher Scientific, catalog number: BP220
    9. HEPES (Fisher Scientific, catalog number: BP310 )
    10. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217 )
    11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Fisher Scientific, catalog number: M33 )
    12. Spermidine (Sigma-Aldrich, catalog number: 85558 )
    13. Dithiothreitol (DTT) (United States Biological, catalog number: D8070 )
    14. cOmplete mini protease inhibitors (Roche Diagnostic, catalog number: 11836153001 )
    15. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
    16. Spermine tetrahydrochloride (Sigma-Aldrich, catalog number: S2876 )
    17. 1x phosphate buffered saline (Thermo Fisher Scientific, GibcoTM, catalog number: 21600069 )
    18. 100 mM EDTA (see Recipes)
    19. 100 mM EGTA (see Recipes)
    20. 2.1 M sucrose solution (see Recipes)
    21. 2.8 M sucrose solution (see Recipes)
    22. Homogenization buffer (see Recipes)
    23. 1% BSA homogenization buffer (see Recipes)
    24. Resuspension buffer (see Recipes)
    25. 1% BSA resuspension buffer (see Recipes)

Equipment

  1. Dissection equipment
    1. Pins (Carolina Biological Supply Company, catalog number: 629122 )
    2. 4.5 inch pointed dissection scissors (Fisher Scientific, catalog number: 08-940 )
    3. Hemostat (GF HEALTH PRODUCTS, catalog number: 2675 )
  2. 15 ml Dounce homogenizer with PTFE serrated plunger (Cole-Parmer Instrument, catalog numbers: 44468-10 and 44468-16 )
  3. UV lamp (optional) (Spectroline, catalog number: ENF-240C )
  4. Centrifuge (Eppendorf, model: 5702 )
  5. Microcentrifuge (International Equipment Company, model: S139068 )
    Note: This product has been discontinued.
  6. Ultracentrifuge (Beckman Coulter, model: OptimaTM LE-80K , LLE7)
  7. SW 32 Ti swing bucket rotor (Beckman Coulter, model: SW 32 Ti , catalog number: 369650)
  8. UltraRocker rocking platform (Bio-Rad Laboratories, catalog number: 1660709EDU )
  9. Magnet (optional) (Thermo Fisher Scientific, catalog number: 12320D )
  10. Round ended microspatula (Fisher Scientific, catalog number: 21-401-5 )

Procedure

Note: All solutions should be chilled to 4 °C and all steps performed at 4 °C to reduce enzyme activity and preserve sample integrity.

  1. Prepare 2.8 M and 2.1 M sucrose solutions (see Recipes) and buffers.
    Note: It is easiest to prepare the sucrose gradient if 8 ml of the 2.8 M sucrose solution is added to the bottom of the 30 ml ultracentrifuge tubes before it is chilled to 4 °C.
  2. Prepare dissection equipment by washing thoroughly with ethanol.
  3. Dissect gastrocnemius and rectus femoris muscles from mouse (Figure 1 and Videos 1 and 2) and place up to 4 muscles in a 10 ml round bottom tube.
    Notes:
    1. The soleus often adheres to the gastrocnemius during dissection. Remove the soleus from the gastrocnemius before proceeding to process the gastrocnemius.
    2. The rectus femoris is one of four muscles that make up the quadriceps.


    Figure 1. Muscle dissection. 1. Starting with the mouse lying on its ventral side, use hemostats to retract skin from ankle to hip. 2. Remove the fat pad over the back of the knee. 3. Insert scissors behind Achilles tendon. 4. Open scissors to separate gastrocnemius muscle from underlying tissue. 5. Cut Achilles tendon and the head of the gastrocnemius. If the soleus is still attached to the gastrocnemius, remove it. 6. Turn the mouse over so it is lying on its back. 7. Remove the fat pad over the hip. 8. With scissors parallel to the femur cut the tendon connecting the rectus femoris to the acetabulum (knee) and continue lengthwise to the hip. Finally, with scissors perpendicular to the femur cut the tendon connecting the muscle to the iliac spine and collect the rectus femoris muscle.

    Video 1. Gastrocnemius dissection. As shown pictorially in Figure 1 steps 1-5, this video demonstrates gastrocnemius dissection.

    Video 2. Rectus femoris dissection. As shown pictorially in Figure 1 steps 6-8, this video demonstrates rectus femoris dissection.

  4. Mince muscles with scissors.
  5. Add 5 ml of chilled homogenization buffer (see Recipes) for 1-2 muscles or 10 ml of chilled homogenization buffer for 3-4 muscles to the minced muscles.
  6. Homogenize muscles with Dounce homogenizer on ice about 50 strokes (until all large chunks of muscle have been forced from the bottom of the homogenizer to the top of the plunger) (Figure 2).
    Notes:
    1. Warning: a vacuum may form between the plunger and the surface of the homogenization buffer. If the plunger rapidly collides the buffer, this can shatter the homogenizer.
    2. If you are pooling homogenate from more than 4 muscles, homogenize them separately and then pool the homogenate prior to Step 7.
    3. Aged or regenerating muscle samples may take longer to homogenize.
    4. Homogenizer and dissection equipment should be cleaned with ethanol and water.


    Figure 2. Pre-ultracentrifugation preparation. 1. Using scissors mince collected muscles in the tube. Intact muscles are shown on the left and minced muscles on the right. 2. Suspend the minced muscles in 10 ml homogenization buffer. 3. Homogenize muscle using a Dounce homogenizer. 4. Pass homogenate through a 40 µm filter into a 50 ml conical tube. Incompletely disrupted tissue and large debris will remain in the filter. 5. Centrifuge the filtrate to obtain a crude nuclear pellet (arrowhead). DAPI clearly labels the nuclear pellet under UV light. Warning: when homogenizing a vacuum may form between the pestle and homogenate. If the pestle is sucked down into the homogenizer, it can strike with sufficient force to shatter the homogenizer.

  7. Rinse a 40 µm filter with 1-2 ml of homogenization buffer. Pass the homogenate over this filter into a 50 ml conical.
    Note: Fibrous material that cannot pass through the filter may clog the filter. Gently scrape the filter with a round ended microspatula to help the filtrate pass through.
  8. Rinse the filter with 5 ml homogenization buffer.
  9. Centrifuge the filtrate at 1,000 x g for 10 min at 4 °C to obtain a crude nuclear pellet.
  10. During the centrifugation, carefully layer 12 ml of the 2.1 M sucrose solution over the 2.8 M sucrose cushion in the 30 ml ultracentrifuge tube.
  11. After the centrifugation is completed, discard the cytoplasmic supernatant and resuspend the crude nuclear pellet in 8 ml homogenization buffer. Carefully layer the resuspended lysate on the top of the sucrose gradient.
    Notes:
    1. If DAPI is added at a final concentration of 1 µg/ml to the homogenate at this step, the position of the nuclei in the sucrose gradient can be visualized.
    2. The cytoplasmic fraction can be saved for subsequent biochemical or proteomic analysis. If used immediately, the cytoplasmic fraction should be kept on ice. For longer term storage the cytoplasmic fraction should be aliquoted and stored at -80 °C.
  12. Balance the ultracentrifuge tubes by weight.
  13. Centrifuge the samples at 186,712 max G (32,000 rpm in SW 32 Ti Beckman swing bucket rotor) for 200 min at 4 °C.
  14. During the centrifugation, coat a 50 ml conical tube with BSA by incubating it with 20 ml 1% BSA resuspension buffer (see Recipes) for at least 30 min at 4 °C with low speed rocking on a standard laboratory rocker.
    Note: Removing the top of a glove box allows the tubes to roll on the rocker resulting in even BSA coating of up to 6 tubes (Figure 3).


    Figure 3. Loading ultracentrifuge tubes. Before beginning the protocol, 2.8 M sucrose should be layered into the bottom of the ultracentrifuge tube and chilled. During 1,000 x g centrifugation step of the muscle homogenate, layer chilled 2.1 M sucrose solution over the 2.8 M solution. When the centrifugation of the muscle homogenate is completed, resuspend the crude nuclear pellet in homogenization buffer and carefully layer it over the 2.1 M solution. DAPI labels the nuclei in the lysate. During the ultracentrifugation step coat 50 ml conical tubes with BSA, which is simplified by allowing the tubes to roll in a box on the rocker.

  15. After the ultracentrifugation is complete, aspirate the upper layers of the sucrose gradient to within a centimeter of the 2.1 M and 2.8 M boundary. Collect the 2.1 M/2.8 M interface into the BSA-coated 50 ml conical tubes using glass Pasteur pipettes (Figure 4 steps 1-4).
    Notes:
    1. If the overlying layers have been correctly removed, there is little danger of contaminating the nuclear fraction with material from another interface. Thus collecting 1 cm above and 1 cm below the interface will increase the yield of nuclei without endangering the purity of the fraction.
    2. If DAPI has been added to the homogenate, the position of the nuclei in the gradient can be visualized with UV light. DAPI fluorescence under UV light can be used to confirm that the entire interface containing the nuclei has been collected (Figure 4 step 4).
    3. Nuclei can be collected by puncturing the side of the ultracentrifugation tube with a 20-gauge needle and 10 ml syringe. However, nuclei are sheared passing through the needle and the high viscosity of the sucrose makes collection difficult. Similarly, collection of fractions beginning with the bottom of the gradient is difficult because of the high viscosity of the 2.8 M sucrose. 


    Figure 4. Unloading ultracentrifuge tubes. 1. Identify the interface between the 2.8 M and 2.1 M sucrose solutions which contains the nuclear fraction (white arrow). It is easy to identify by DAPI fluorescence under UV light. 2. Aspirate the overlying layers to a centimeter above the nuclear fraction (white arrow). 3. Collect the nuclear fraction into the BSA-coated 50 ml conical tube. Nuclei in the fraction can be observed by DAPI fluorescence. 4. After collecting the nuclear fraction, the thin gray band of nuclei will no longer be visible and no DAPI fluorescence will be detectable in the ultracentrifuge tube or sucrose collected from the surface of the remaining solution. 5. Resuspend the collected nuclear fraction 1:10 in resuspension buffer by inverting the BSA-coated 50 ml conical tube several times. 6. Pellet the purified nuclei by centrifugation. The nuclear pellet is easy to see both with and without DAPI labeling. 7. Remove the supernatant and resuspend the pellet in 1.5 ml resuspension buffer. 8. Pellet the nuclei by centrifugation. Nuclei can be resuspended for imaging or washed 2 more times for biochemical and proteomic analyses.

  16. Dilute the collected interface in the 50 ml conical tube 1:10 with resuspension buffer (see Recipes). Mix by inverting 5-10 times (Figure 4 step 5).
    Note: The collected interface is much denser than the resuspension buffer. Be careful to completely mix the interface and resuspension buffer.
  17. Centrifuge the diluted interface in the 50 ml conical tube at 1,000 x g for 10 min at 4 °C (Figure 4 step 6).
  18. Discard the supernatant and resuspend the nuclear pellet in 1 ml resuspension buffer and transfer to a 1.7 ml tube.
    Note: For flow cytometry and microscopy, resuspend the nuclei in chilled resuspension buffer with 1% BSA. 
  19. Centrifuge the resuspended nuclei at 800 x g for 10 min at 4 °C (Figure 4 step 8). 
  20. Discard the supernatant and resuspend the nuclear pellet in appropriate buffer (see Figure 5A for representative images of isolated nuclei).
    Notes:
    1. For flow cytometry, resuspend the nuclear pellet in 500 µl resuspension buffer with 1% BSA and pass through a 35 µm cell strainer into a 5 ml polystyrene tube before analyzing. 
    2. For microscopy, resuspend the nuclear pellet in 1-3 ml resuspension buffer with 1% BSA.
    3. For Western blot or mass spectrometry, wash the nuclear pellet two more times with resuspension buffer to remove residual BSA, then resuspend in an appropriate denaturing buffer. 

Optional alternative approach
If biochemical purity is not critical, nuclei can be isolated more rapidly by affinity purification. Nuclei isolated by affinity purification are depleted for mitochondrial and cytoplasmic markers but retain substantial amounts of endoplasmic reticulum markers. If specific nuclear markers are known, affinity-based purification can be easily applied to other cell types (Deal and Henikoff, 2011).
Note: All solutions should be chilled to 4 °C and all steps performed at 4 °C.

  1. Wash 1.5 mg Protein A-conjugated magnetic beads by incubating with 500 µl homogenization buffer in a 1.7 ml microcentrifuge tube for 5 min with rocking. Then place the tube in a magnetic separation rack and wait 20 sec for the beads to accumulate on the side of the tube and aspirate the buffer. Repeat 2 times.
  2. Add 5 µg anti-TMEM38A antibody and 200 µl homogenization buffer with 1% BSA to the washed beads. Incubate at 4 °C with low speed rocking for 30 min.
    Note: Magnetic beads rather than agarose or sepharose beads must be used because debris will cosediment with the agarose or sepharose beads but can be separated from magnetic beads which accumulate on the side of the tube where the magnet is placed.
  3. Process samples as described above for Steps 2-8.
  4. After centrifugation to obtain a crude nuclear pellet (described in Step 8 above), discard the supernatant and resuspend the pellet in 1 ml chilled homogenization buffer with 1% BSA.
  5. Incubate the magnetic beads with the resuspended nuclei from up to 4 muscles at 4 °C for 30 min with gentle rocking.
  6. Place the tubes containing the beads in a magnetic separation rack and wait 20 sec for the beads to accumulate on the side of the tube. Aspirate the buffer and debris at the bottom of the tube.
  7. Wash the magnetic beads once with 500 µl chilled homogenization buffer with 1% BSA (see Recipes).
  8. Resuspend the beads in an appropriate buffer for the downstream application (see Figure 5B for representative images of isolated nuclei).
    Notes:
    1. For flow cytometry, resuspend the nuclei in 500 µl resuspension buffer with 1% BSA and pass through a 35 µm cell strainer into a 5 ml polystyrene tube before analyzing.
    2. For microscopy, resuspend the nuclei in resuspension buffer with 1% BSA.
    3. For Western blot or mass spectrometry, wash the nuclei twice with PBS to remove residual BSA and resuspend in the appropriate denaturing buffer for downstream application. 


    Figure 5. Purified myonuclei. A. Nuclei isolated by ultracentrifugation through 2.1 M sucrose. Nuclei are visible in DAPI channel. B. Affinity purified nuclei isolated by binding of TMEM38A antibody and magnetic beads to nuclei. Nuclei are visible in DAPI channel. Scale bar = 10 µm.

Data analysis

  1. Data should be processed according to standard practice for the relevant downstream technique. Appropriate downstream analysis can include microscopy, Western blot, proteomics, and flow cytometry.
  2. The average yield from a single mouse gastrocnemius muscle is 1 x 106 nuclei. Pooling two gastrocnemius and two rectus femoris muscles yields 6 x 106 nuclei and 8 µg of total protein. Isolating nuclei from the tibialis anterior muscle resulted in a very low yield of nuclei that was impractical to analyze. It should be noted that unless care is taken to wash nuclei thoroughly, residual BSA can artificially inflate measurements of total protein. For this reason, it is recommended to first check purity and yield by microscopy and then measure protein concentration if applicable. The yield and purity are consistent between experiments. The greatest variation in yield resulted from failure to keep samples at 4 °C for the duration of the experiment.
  3. For proteomics applications, we had the greatest success when both gastrocnemius and rectus femoris muscles from 3 mice were pooled. Proteomics experiments should be completed with at least 5 biological replicates to detect small changes in protein abundance.
  4. For analysis by flow cytometry nuclei isolated from a single gastrocnemius or rectus femoris muscle provided ample starting material. Flow cytometry experiments should include at least three biological replicates.

Notes

  1. The biochemical purification of myonuclei requires about 5 h to complete. If a large number of samples are being prepared, more time is required. Magnetic bead affinity based preparation requires about 1 h to complete.
  2. Endoplasmic reticulum depletion is variable across preparations.

Recipes

  1. 100 mM EDTA (50 ml)
    Combine:
    372 mg EDTA
    40 ml ultra-pure water
    Adjust pH to 7 with NaOH
    Adjust final volume to 50 ml with ultra-pure water
    Store at room temperature for up to a year
  2. 100 mM EGTA (50 ml)
    Combine:
    1.9 g EGTA
    40 ml ultra-pure water
    Adjust to pH 8 with NaOH
    Adjust final volume to 50 ml with ultra-pure water
    Store at room temperature for up to a year
  3. 2.1 M sucrose solution (50 ml)
    Combine the following over high heat with medium stirring:
    35.9 g sucrose
    2.5 ml HEPES (1 M)
    1.25 ml KCl (1 M)
    2.5 ml MgCl2 (100 mM)
    Ultra-pure water to 50 ml
    Note: Because the solution is made over heat, some of the fluid will boil off. Adjust the final volume after the solution has been removed from heat.
    Store at 4 °C for up to 1 week
  4. 2.8 M sucrose solution (25 ml)
    Combine the following over high heat with medium stirring:
    24 g sucrose
    1.25 ml HEPES (1 M)
    625 µl KCl (1 M)
    1.25 ml MgCl2 (100 mM)
    Ultra-pure water to 25 ml
    Note: Because the solution is made over heat, some of the fluid will boil off. Adjust the final volume after the solution has been removed from heat.
    Prepare fresh for each use
  5. Homogenization buffer (50 ml)
    Combine the following:
    500 µl HEPES (1 M)
    3 ml KCl (1 M)
    250 µl spermidine (100 mM)
    750 µl spermine tetrahydrochloride (10 mM)
    10 ml EDTA (10 mM)
    250 µl EGTA (100 mM)
    2.5 ml MgCl2 (100 mM)
    5.13 g sucrose
    Ultra-pure water to 50 ml
    Filter sterilize and store at 4 °C for up to 1 month
    Immediately prior to use add:
    100 µl DTT (1 M)
    5 Roche cOmplete mini protease inhibitor tablets
  6. 1% BSA homogenization buffer (50 ml)
    Combine the following:
    500 mg BSA
    50 ml homogenization buffer
    Prepare fresh for each use
  7. Resuspension buffer (50 ml)
    Combine the following:
    1 ml HEPES (1 M)
    750 µl MgCl2 (100 mM)
    500 µl KCl (1 M)
    250 µl spermidine (100 mM)
    750 µl spermine tetrahydrochloride (10 mM)
    1 ml EDTA (10 mM)
    45.75 ml ultra-pure water
    Store at 4 °C for up to 1 month
  8. 1% BSA resuspension buffer (50 ml)
    Combine the following:
    500 mg BSA
    50 ml resuspension buffer
    Prepare fresh for each use

Acknowledgments

This work was supported by grants to GKP (AR062483), to AAC (AR067645), and a training grant (T32GM008367) from the National Institutes of Health. The described protocol was adapted from the work of Wilkie and Schirmer (2008). No potential conflict of interest was reported by the authors.

References

  1. Bleunven, C., Treves, S., Xia Jinyu, X., Leo, E., Ronjat, M., De Waard, M., Kern, G., Flucher, B. E. and Zorzato, F. (2008). SRP-27 is a novel component of the supramolecular signalling complex involved in skeletal muscle excitation–contraction coupling. Biochem J 411(2): 343-349.
  2. Cutler, A. A., Dammer, E. B., Doung, D. M., Seyfried, N. T., Corbett, A. H. and Pavlath, G. K. (2017). Biochemical isolation of myonuclei employed to define changes to the myonuclear proteome that occur with aging. Aging Cell 16(4): 738-749.
  3. Deal, R. B. and Henikoff, S. (2011). The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6(1): 56-68.
  4. Deshmukh, A. S., Murgia, M., Nagaraj, N., Treebak, J. T., Cox, J. and Mann, M. (2015). Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors. Mol Cell Proteomics 14(4): 841-853.
  5. Dimauro, I., Pearson, T., Caporossi, D. and Jackson, M. J. (2012). A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue. BMC Res Notes 5: 513.
  6. Jankowska, U., Latosinska, A., Skupien-Rabian, B., Swiderska, B., Dziedzicka-Wasylewska, M. and Kedracka-Krok, S. (2016). Optimized procedure of extraction, purification and proteomic analysis of nuclear proteins from mouse brain. J Neurosci Methods 261: 1-9.
  7. Ohkawa, Y., Mallappa, C., Vallaster, C. S. and Imbalzano, A. N. (2012). Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays. Methods Mol Biol 798: 517-530.
  8. Wilkie, G. S. and Schirmer, E. C. (2008). Purification of nuclei and preparation of nuclear envelopes from skeletal muscle. Methods Mol Biol 463: 23-41.

简介

骨骼肌提供运动,吞咽和呼吸所需的收缩力,因此是生存所必需的。骨骼肌细胞是独特的,因为它们是含有数千个核的极大细胞。这些细胞核必须协调一致才能维持骨骼肌功能,从而维持生命。细胞核是信号整合和基因表达调控的主要部位。然而,检查骨骼肌中的核过程可能是困难的,因为肌核难以分离。我们优化了一个协议,从整个肌肉组织使用超速离心蔗糖梯度来分离核分数的纯化肌细胞。我们将这些纯化的细胞核用于下游应用,包括流式细胞术和质谱。我们使用这种方法来比较年轻和老鼠后肢肌肉的肌细胞蛋白质组(Cutler等人,2017)。这个协议可能适用于分离myonuclei的各种下游分析,如流式细胞仪,显微镜,蛋白质印迹和蛋白质组学。

【背景】为了生存,必须保持适当的骨骼肌功能。这种维持的一个组成部分是根据细胞需要和环境线索调整基因表达。调节基因表达的核过程是调节细胞组成和行为的关键组成部分。然而,由于四个技术限制,涉及这些过程的肌细胞蛋白难以研究。首先,骨骼肌是致密的,紧密地填满了组织中超过60%的蛋白质的收缩性蛋白质(Deshmukh等人,2015; Cutler等人)。 ,2017)。这些高丰度的收缩性蛋白质消除了不那么丰富的核蛋白质。其次,骨骼肌的致密纤维结构使得难以解离而不损伤核,使得核难以分离。第三,离心后的密集的碎片与细胞核结合,从组织分离核的困难。第四,骨骼肌由多种细胞类型组成,因此分离的核和检测到的核蛋白可能来自其他细胞类型的肌核或细胞核。

已经优化了几种方法来丰富不同生物体的肌细胞以用于各种下游应用。大川等。提出了用于从小鼠组织中分离肌细胞的详细方案,其被开发用于最大化交联试剂用于染色质免疫沉淀(ChIP)分析(Ohkawa et al。,2012)。 Wilkie和Shrimer开发了一种分离肌红蛋白包膜和肌浆网以进行蛋白质组比较的方法(Wilkie和Schirmer,2008)。一种由Dimauro等人优化的方法。同时收集线粒体,细胞核和细胞质部分以比较不同细胞区室中的蛋白质定位(Dimauro et al。,2012)。尽管这些从骨骼肌组织中富集核的方法对预期的随后的分析都是有效的,但是它们没有优先分离完整的核并且不区分来自其他细胞类型的肌核和细胞核。在Arabidopsis thaliana(Deal and Henikoff,2011)中开发了一种基于亲和力的方法来选择性地从特定细胞类型中分离细胞核,现在可用于小鼠(Jankowska et al。 )。 ,2016)。然而,这种基于亲和力的方法需要对感兴趣的细胞类型进行遗传标记,这使得检查来自多个小鼠模型的肌细胞难以进行麻烦。我们优化了超速离心蔗糖梯度为基础的分馏方法,需要相对较小的样本量,没有遗传标记,并与流式细胞仪和质谱法下游分析兼容。分离的核是完整的,从非核细胞器生物化学地消耗蛋白质,并且85%的核是肌核。为了更快速地分离肌红细胞,我们还使用肌细胞核特异性核包膜蛋白跨膜蛋白38A(TMEM38A)(Bleunven等人,2008; Cutler等人, ,2017)和磁珠。这种方法更快速,产生具有可比较的群体纯度的核,以通过超速离心进行核分离,但具有较低的生物化学纯度。这些核分离技术可用于纯化来自任何小鼠模型的肌细胞以用于不同的下游分析。


关键字:分馏, 蔗糖梯度, 磁分离, 超速离心

材料和试剂

  1. 物料
    1. 30毫升ultraclear ultracentrifuge管(贝克曼库尔特,目录号码:344058)
    2. 10ml圆底聚丙烯管(Corning,Falcon,目录号:352059)
    3. 40微米尼龙网格细胞过滤器(Biologix,目录号:15-1040)
    4. 50ml锥形聚丙烯管(BioExpress,目录号:C-3394-4)
    5. 15厘米玻璃巴斯德吸管(Fisher Scientific,目录号:13-678-20A)
    6. 20号针(Fisher Scientific,目录号:14-826-5C)
      制造商:BD,目录号:305176。
    7. 10毫升注射器(BD,目录号:309604)
    8. 1.7 ml低粘性疏水管(BioExpress,GeneMate,目录号:C-3302-1)
    9. 5毫升锥形聚苯乙烯管与细胞过滤器盖(康宁,目录号:352235)
    10. 0.45μm聚丙烯注射器过滤器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:F2500-9)

  2. 试剂
    1. 乙醇
    2. 4,6-二脒基-2-苯基吲哚(DAPI)(Sigma-Aldrich,目录号:D9542)
    3. 蛋白A-缀合的磁珠(Thermo Fisher Scientific,Invitrogen TM,目录号:10001D)
    4. 抗TMEM38A抗体(Merck,目录号:06-1005)
    5. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:BP120)
    6. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318)
    7. 乙二醇 - 双(β-氨基乙基醚)N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
    8. 蔗糖(Fisher Scientific,目录号:BP220) 
    9. HEPES(Fisher Scientific,目录号:BP310)
    10. 氯化钾(KCl)(Fisher Scientific,目录号:P217)
    11. 氯化镁六水合物(MgCl 2•6H 2 O)(Fisher Scientific,目录号:M33)
    12. 亚精胺(Sigma-Aldrich,目录号:85558)
    13. 二硫苏糖醇(DTT)(美国生物,目录号:D8070)
    14. 完全小型蛋白酶抑制剂(Roche Diagnostic,目录号:11836153001)
    15. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
    16. 精胺四盐酸盐(Sigma-Aldrich,目录号:S2876)
    17. 1x磷酸盐缓冲盐水(Thermo Fisher Scientific,Gibco TM,目录号:21600069)
    18. 100 mM EDTA(见食谱)
    19. 100 mM EGTA(见食谱)
    20. 2.1 M蔗糖溶液(见食谱)
    21. 2.8 M蔗糖溶液(见食谱)
    22. 匀浆缓冲液(见食谱)
    23. 1%BSA均质缓冲液(见食谱)
    24. 重悬缓冲液(见食谱)
    25. 1%BSA重悬缓冲液(见食谱)

设备

  1. 解剖设备
    1. 针(卡罗莱纳州生物供应公司,目录号:629122)
    2. 4.5寸尖头解剖剪(Fisher Scientific,目录号:08-940)
    3. 止血剂(GF HEALTH PRODUCTS,目录号:2675)
  2. 15毫升Dounce均化器与聚四氟乙烯锯齿柱塞(科尔 - 帕默仪器,目录号码:44468 - 10和44468 - 16)
  3. 紫外灯(可选)(Spectroline,目录号:ENF-240C)
  4. 离心机(Eppendorf,型号:5702)
  5. 微量离心机(国际设备公司,型号:S139068)
    注:此产品已停产。
  6. 超速离心机(Beckman Coulter,型号:Optima TM LE-80K,LLE7)
  7. SW 32钛回转斗式转子(Beckman Coulter,型号:SW 32 Ti,目录号:369650)
  8. UltraRocker摇摆平台(Bio-Rad Laboratories,目录号:1660709EDU)
  9. 磁铁(可选)(Thermo Fisher Scientific,目录号:12320D)
  10. 圆形的微小支架(Fisher Scientific,目录号:21-401-5)

程序

注意:所有的溶液都应该冷冻到4°C,所有的步骤都要在4°C下进行,以降低酶的活性,保持样品的完整性。

  1. 准备2.8 M和2.1 M蔗糖溶液(见食谱)和缓冲液。
    注意:如果将8ml的2.8M蔗糖溶液加入到30ml超速离心管的底部,然后将其冷却至4℃,则制备蔗糖梯度是最容易的。

  2. 用乙醇彻底清洗切碎设备。
  3. 解剖小鼠的腓肠肌和股直肌(图1和视频1和2),并在10 ml圆底管中放置4个肌肉。
    注意:
    1. 比目鱼肌在解剖过程中经常粘附在腓肠肌上。
      在进行腓肠肌处理之前,从腓肠肌取出比目鱼。
    2. 股直肌是组成四头肌的四块肌肉之一。


    图1.肌肉解剖 1.从鼠标躺在腹侧开始,使用止血钳将皮肤从脚踝收回到臀部。 2.移除膝盖后面的脂肪垫。 3.将剪刀插入跟腱后面。 4.用剪刀将腓肠肌与下面的组织分开。 5.切开跟腱和腓肠肌的头部。如果比目鱼仍附着在腓肠肌上,请将其取出。 6.将鼠标转过来,使其仰卧。 7.取下臀部的脂肪垫。用平行于股骨的剪刀剪断将股直肌连接至髋臼(膝盖)的肌腱并纵向延伸至髋部。最后,用垂直于股骨的剪刀剪断将肌肉连接到髂骨的肌腱并收集股直肌。

    视频1

    视频2
  4. 用剪刀剁碎肌肉。

  5. 加入5ml冷冻均质缓冲液(参见食谱)1-2个肌肉或10ml冷却的均化缓冲液,使3-4个肌肉达到切碎的肌肉。
  6. 用Dounce匀浆器在冰上匀浆约50次(直到所有的大块肌肉都从匀浆器的底部被压到柱塞的顶部)(图2)。
    注意:
    1. 警告:活塞与均质缓冲器表面之间可能会形成真空。如果柱塞快速撞击缓冲液,可能会破碎均质器。
    2. 如果你从4个以上的肌肉匀浆匀浆,分别匀浆,然后在步骤7之前将匀浆混合。
    3. 老年人或再生肌肉样本可能需要更长的时间才能均匀。
    4. 均质器和解剖器械应该用乙醇和水清洗。


    图2.预超速离心制备1.使用剪刀收集管中收集的肌肉。完整的肌肉显示在左侧,右侧显示肌肉。 2.将绞碎的肌肉悬浮在10毫升均化缓冲液中。 3.使用Dounce匀浆器匀化肌肉。 4.将匀浆通过40μm过滤器,通入50ml锥形管中。不完全破坏的组织和大碎片将保留在过滤器。 5.将滤液离心以获得粗制的核颗粒(箭头)。 DAPI在UV光下清楚地标记核小球。 警告:杵和匀浆之间可能形成均质真空。如果杵被吸入均化器,它可以用足够的力量击碎均化器。

  7. 用1-2毫升匀浆缓冲液冲洗一个40微米的过滤器。通过这个过滤器的匀浆到50毫升圆锥形。
    注意:不能通过过滤器的纤维材料可能会堵塞过滤器。

    轻轻地刮过滤器,用一个圆形的微小支架来帮助滤液通过。
  8. 用5毫升匀浆缓冲液冲洗过滤器。
  9. 将滤液在4℃下1000×g离心10分钟以获得粗制的核颗粒。
  10. 在离心过程中,在30ml超速离心管中,将2.8ml 2.1M蔗糖溶液小心地置于2.8M蔗糖垫上。
  11. 离心完成后,弃去细胞质上清液,并在8毫升均质缓冲液中重悬粗核沉淀。小心地将重悬的裂解物铺在蔗糖梯度的顶部。
    注意:
    1. 如果在此步骤中将DAPI以1μg/ ml的终浓度添加到匀浆中,则可以显现蔗糖梯度中的细胞核的位置。
    2. 可以保存细胞质部分用于随后的生化或蛋白质组分析。如果立即使用,细胞质部分应保存在冰上。为了长期储存,应将细胞质部分分装并储存在-80℃。
  12. 按重量平衡超速离心管。
  13. 在4°C下,将样品以186,712 G(SW 32 Ti Beckman摆动转子中的32,000 rpm)离心200分钟。
  14. 在离心过程中,通过在20℃下用20ml 1%BSA重悬缓冲液(参见食谱)在4℃下用标准实验室摇床低速摇动至少30分钟,涂布含有BSA的50ml锥形管。
    注意:取下手套箱的顶部可以使管子在摇杆上滚动,甚至可以将多达6个试管的BSA涂层(图3)。


    图3.加载超速离心管。在开始实验之前,应将2.8 M蔗糖分层放入超速离心管底部并冷冻。在肌肉匀浆的1,000×g离心步骤期间,在2.8M溶液上层冷却2.1M蔗糖溶液。完成肌匀浆的离心分离后,将粗核沉淀物在匀浆缓冲液中重新悬浮,并小心地在2.1M溶液上铺满。 DAPI标记裂解物中的细胞核。在超离心步骤期间涂布50毫升含有BSA的锥形管,通过允许管在摇杆上的盒子中滚动来简化。
  15. 超速离心完成后,将上层蔗糖梯度抽吸到2.1M和2.8M边界的厘米内。使用玻璃巴斯德吸管收集2.1 M / 2.8 M接口到BSA涂层的50 ml锥形管中(图4步骤1-4)。
    注意:
    1. 如果上覆层已被正确地移除,那么用来自另一界面的材料污染核碎片的危险很小。因此,在界面以上1厘米和1厘米处收集将增加核的产率而不危害该分数的纯度。
    2. 如果已将DAPI添加到匀浆中,则可以用紫外光使梯度中的细胞核的位置可视化。在紫外光下的DAPI荧光可以用来确认收集了包含细胞核的整个界面(图4步骤4)。
    3. 可以通过用20号针头和10ml注射器刺穿超速离心管的一侧来收集细胞核。但是,细胞核被剪切穿过针头,蔗糖的高粘度使得收集困难。类似地,由于2.8M蔗糖的高粘度,难以从梯度底部开始收集馏分。 


    图4.卸载超速离心管1.确定含有核分数的2.8M和2.1M蔗糖溶液之间的界面(白色箭头)。通过DAPI荧光在紫外光下很容易识别。 2.将覆盖层吸入核分数以上一厘米(白色箭头)。 3.将核分数收集到BSA包被的50ml锥形管中。可以通过DAPI荧光观察到部分中的核。 4.收集细胞核组分后,细胞核的薄的灰色带将不再可见,在超速离心管或从剩余溶液表面收集的蔗糖中不能检测到DAPI荧光。 5.通过将BSA包被的50ml锥形管颠倒几次,将悬浮于悬浮缓冲液中的收集的细胞核分数1:10重新悬浮。 6.通过离心沉淀纯化的细胞核。无论是否使用DAPI标记,核颗粒都很容易看到。 7.取出上清液,并重悬在1.5毫升悬浮缓冲液沉淀。 8.离心沉淀细胞核。核可以重新悬浮成像或洗2次以上的生化和蛋白质组学分析。

  16. 稀释收集的界面在50毫升锥形管1:10用重悬缓冲液(见食谱)。
    反转5-10次(图4步骤5) 注意:所收集的接口比重悬缓冲区更密集。小心完全混合界面和重悬缓冲区。
  17. 在4℃下将稀释的界面在1,000ml xg的1000ml锥形管中离心10分钟(图4步骤6)。
  18. 弃去上清液,重新悬浮在1毫升悬浮缓冲液核颗粒,并转移到1.7毫升管。
    注意:对于流式细胞术和显微镜检查,用1%BSA将细胞核悬浮在冷冻的重悬缓冲液中。
  19. 将重新悬浮的细胞核在4℃以800×g离心10分钟(图4步骤8)。 
  20. 丢弃上清液,并在适当的缓冲液中重新悬浮核沉淀(见图5A代表孤立核的代表性图像)。
    注意:
    1. 对于流式细胞术,用1%BSA重悬沉淀于500μl重悬缓冲液中,并在分析之前通过35μm细胞过滤器进入5ml聚苯乙烯管。< em>
    2. 对于显微镜检查,用1%BSA重新悬浮于1-3ml重悬浮缓冲液中的核沉淀。
    3. 对于蛋白质印迹或质谱法,用再悬浮缓冲液洗两次核沉淀以去除残余的BSA,然后重悬于合适的变性缓冲液中。< em>

可选替代方法
如果生化纯度不重要,可以通过亲和纯化更快地分离核。通过亲和纯化分离的核耗尽线粒体和细胞质标记,但保留大量的内质网标记。如果已知特定的核标记,亲和纯化可以很容易地应用于其他细胞类型(Deal和Henikoff,2011)。
注意:所有溶液应该冷冻到4°C,所有的步骤都要在4°C下进行。

  1. 通过在1.7ml微量离心管中用500μl匀浆缓冲液孵育5分钟,摇动1.5mg蛋白A缀合的磁珠。然后将试管置于磁力分离架上等待20秒,使试剂珠在试管侧积聚并吸取缓冲液。重复2次。
  2. 加入5微克抗TMEM38A抗体和200微升含有1%BSA的均化缓冲液。在4°C下低温摇动30分钟。
    注意:必须使用磁珠而不是琼脂糖或琼脂糖珠,因为碎片会与琼脂糖或琼脂糖珠结合,但可以与磁珠堆积在放置磁体的管侧面的磁珠分离。
  3. 如上述步骤2-8所述处理样品。
  4. 离心得到粗制的核沉淀(在上述步骤8中描述)后,丢弃上清液并将沉淀重悬于1ml含有1%BSA的冷冻均质缓冲液中。
  5. 孵育磁珠与重新悬浮的细胞核从4肌肉在4°C的30分钟轻轻摇摆。
  6. 将包含珠子的试管置于磁力分离架上,等待20秒,使珠子积聚在试管的侧面。
    吸入管底部的缓冲液和碎屑
  7. 用500μl含1%BSA的冷冻均质缓冲液清洗磁珠一次(见食谱)。

  8. 在下游应用中将珠子重悬在合适的缓冲液中(见图5B) 注意:
    1. 对于流式细胞术,用1%BSA将细胞核重新悬浮于500μl重悬缓冲液中,并在分析前通过35μm细胞过滤器进入5ml聚苯乙烯管中。
    2. 对于显微镜检查,用1%BSA重悬悬浮缓冲液中的核。
    3. 对于蛋白质印迹或质谱法,用PBS洗涤细胞核两次以去除残留的BSA,并重悬于适当的变性缓冲液中用于下游应用。< / em>


    图5.纯化的肌细胞核A.通过2.1M蔗糖超速离心分离的核。核在DAPI通道中可见。 B.通过将TMEM38A抗体和磁珠与核结合而分离的亲和纯化的核。核在DAPI通道中可见。比例尺= 10微米。

数据分析

  1. 数据应根据相关下游技术的标准实践进行处理。适当的下游分析可以包括显微镜,蛋白质印迹,蛋白质组学和流式细胞仪。
  2. 来自单个小鼠腓肠肌的平均产量是1×10 6个核。将两个腓肠肌和两个股直肌集中,产生6×10 6个核和8μg总蛋白质。从胫骨前肌分离核导致核的产量非常低,这是不切实际的分析。应该注意的是,除非注意彻底清洗细胞核,否则残余的BSA可能会人为地膨胀总蛋白的测量值。为此,建议首先通过显微镜检查纯度和产量,然后测量蛋白质浓度(如果适用)。产率和纯度在实验之间是一致的。产量变化最大的原因是在实验过程中未能将样品保持在4°C。
  3. 对于蛋白质组学应用,我们取得了最大的成功,当三只小鼠的腓肠肌和股直肌都汇集起来。蛋白质组学实验应完成至少5个生物学重复,以检测蛋白质丰度的小变化。
  4. 为了通过流式细胞术进行分析,从单个腓肠肌或股直肌分离的核提供充足的起始材料。流式细胞术实验应该包括至少三个生物学重复。

笔记

  1. 肌红蛋白的生化纯化需要约5小时才能完成。如果准备大量样本,则需要更多的时间。基于磁珠亲和的制备需要约1小时才能完成。
  2. 内质网耗尽在各制剂中是可变的。

食谱

  1. 100毫克EDTA(50毫升)
    合并:
    372毫克EDTA
    40毫升超纯水
    用NaOH调节pH值至7 用超纯水调节终体积至50毫升
    在室温下储存长达一年
  2. 100毫升EGTA(50毫升)
    合并:
    1.9克EGTA
    40毫升超纯水
    用NaOH调节pH值至8 用超纯水调节终体积至50毫升
    在室温下储存长达一年
  3. 2.1 M蔗糖溶液(50毫升)

    在高温和中等搅拌下结合以下几点: 35.9克蔗糖
    2.5毫升HEPES(1M)
    1.25毫升氯化钾(1M)
    2.5毫升MgCl 2(100毫摩尔)
    超纯水至50毫升
    注意:由于溶液是通过加热,所以一些流体会沸腾。








    在4°C储存长达一周
  4. 2.8 M蔗糖溶液(25毫升)

    在高温和中等搅拌下结合以下几点: 24克蔗糖
    1.25毫升HEPES(1M)
    625微升KCl(1 M)
    1.25毫升MgCl 2(100毫摩尔)
    超纯水至25毫升
    注意:由于溶液是通过加热,所以一些流体会沸腾。







    准备新鲜的每个使用
  5. 匀浆缓冲液(50毫升)
    结合以下内容:
    500μlHEPES(1 M)
    3毫升氯化钾(1M)
    250μl亚精胺(100 mM)
    750μl精胺四氢氯化物(10mM)
    10毫升EDTA(10毫米)
    250μlEGTA(100 mM)
    2.5毫升MgCl 2(100毫摩尔)
    5.13克蔗糖
    超纯水至50毫升
    过滤消毒,并在4°C储存长达1个月
    在使用之前立即添加:
    100μlDTT(1 M)
    5罗氏cOmplete迷你蛋白酶抑制剂片
  6. 1%BSA均质缓冲液(50毫升)
    结合以下内容:
    500毫克BSA
    50毫升均化缓冲液
    准备新鲜的每个使用
  7. 重悬缓冲液(50毫升)
    结合以下内容:
    1毫升HEPES(1M)
    750μlMgCl 2(100mM)
    500微升氯化钾(1 M)
    250μl亚精胺(100 mM)
    750μl精胺四氢氯化物(10mM)
    1毫升EDTA(10毫米)
    45.75毫升超纯水
    在4°C储存长达1个月
  8. 1%BSA重悬缓冲液(50毫升)
    结合以下内容:
    500毫克BSA
    50毫升重悬缓冲液
    准备新鲜的每个使用

致谢

这项工作得到了GKP(AR062483),AAC(AR067645)和美国国立卫生研究院的培训资金(T32GM008367)的资助。所描述的协议是从Wilkie和Schirmer(2008)的工作改编的。提交人没有报告潜在的利益冲突。

参考

  1. Bleunven,C.,Treves,S.,Xia Jinyu,X.,Leo,E.,Ronjat,M.,De Waard,M.,Kern,G.,Flucher,B.E。和Zorzato,F.(2008)。 SRP-27是涉及骨骼肌兴奋 - 收缩偶联的超分子信号复合物的新颖组分。生物化学杂志411(2):343-349。
  2. Cutler,A.A.,Dammer,E.B。,Doung,D.M.,Seyfried,N.T.,Corbett,A.H。和Pavlath,G.K。(2017)。 肌红蛋白的生物化学分离,用于定义伴随衰老发生的肌细胞蛋白质组的变化。老化细胞 16(4):738-749。
  3. Deal,R.B.和Henikoff,S.(2011)。 在拟南芥中进行细胞类型特异性基因表达和染色质分析的INTACT方法 em>。 Nat Protoc 6(1):56-68。
  4. Deshmukh,A. S.,Murgia,M.,Nagaraj,N.,Treebak,J. T.,Cox,J.和Mann,M。(2015)。 小鼠骨骼肌的深层蛋白质组学可以定量蛋白质亚型,代谢途径和转录因子。 Mol Cell Proteomics 14(4):841-853。
  5. Dimauro,I.,Pearson,T.,Caporossi,D。和Jackson,M.J。(2012)。 一种用于骨骼肌细胞和组织的亚细胞分级的简单方案 BMC Res Notes 5:513.
  6. Jankowska,U.,Latosinska,A.,Skupien-Rabian,B.,Swiderska,B.,Dziedzicka-Wasylewska,M.和Kedracka-Krok,S.(2016)。 优化的小鼠脑核蛋白提取,纯化和蛋白质组学分析程序 < J Neurosci Methods 261:1-9。
  7. Ohkawa,Y.,Mallappa,C.,Vallaster,C.S。和Imbalzano,A.N。(2012)。 从骨骼肌卫星细胞和肌纤维分离细胞核,用于染色质免疫沉淀实验。方法Mol Biol 798:517-530。
  8. Wilkie,G.S。和Schirmer,E.C。(2008)。 细胞核的提纯和骨骼肌细胞核的制备 Methods Mol Biol 463:23-41。
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引用:Cutler, A. A., Corbett, A. H. and Pavlath, G. K. (2017). Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue. Bio-protocol 7(24): e2654. DOI: 10.21769/BioProtoc.2654.
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