搜索

Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
从培养的哺乳动物细胞中差异盐分馏核分析染色质相关蛋白   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. The extent of association with chromatin changes rapidly in response to stresses, such as immune activation, oxidative stress, or viral infection, resulting in downstream effects on chromatin conformation and transcription of target genes. To elucidate changes in the composition of proteins associated with chromatin under different conditions, we adapted existing protocols to isolate nuclei and fractionate cellular chromatin using a gradient of salt concentrations. The presence of specific proteins in different salt fractions can be assessed by Western blotting or mass spectrometry, providing insight into the degree to which they are associated with chromatin.

Keywords: Chromatin(染色质), Fractionation(分馏), Salt gradient(盐梯度), Virus(病毒), Chromatin association(染色质结合), Micrococcal nuclease(微球菌核酸酶)

Background

Many chromatin-associated proteins are insoluble under low salt conditions because of their charged-based interaction with DNA or histones. Since salt disrupts charged-based protein-DNA and protein-protein interactions, chromatin-associated proteins become more soluble with increasing concentration of NaCl (Teves and Henikoff, 2012). Proteins strongly bound to DNA are expected to elute with high salt whereas loosely bound proteins, such as transcription factors, will elute with low salt. We are specifically interested in how virus infection alters the composition of factors associated with the cellular chromatin. Nuclear replicating viruses, such as adenovirus, herpes simplex virus, and Epstein-Barr virus, dramatically alter the appearance of the host chromatin during infection (Avgousti et al., 2016; Lam et al., 2010; Simpson-Holley et al., 2005; Chiu et al., 2013). We hypothesized that these changes in appearance are partly due to differences in protein composition of host chromatin. Changes in host chromatin could reflect antiviral defenses mounted by the cell or active manipulation by the virus. To compare association of proteins with chromatin in uninfected and infected cells we developed this protocol to fractionate nuclei using a salt gradient (Figure 1). In this protocol we isolate nuclei, digest the DNA down to mono-nucleosome length, and then wash the nuclei with increasing concentrations of salt, collecting each fraction for analysis by Western blotting. We recently used this protocol to elucidate changes to cellular chromatin during infection with adenovirus (Avgousti et al., 2016). We now present this protocol as a general approach to monitor association of proteins with chromatin under a wide range of perturbing conditions.


Figure 1. Schematic of nuclear fractionation and example Western blot. A. Roughly 4 x 107 cells are prepared per condition. B. Plasma membranes are permeabilized and nuclei are isolated either by sucrose cushion (step B1) or using a Dounce homogenizer (step B2). C. DNA is digested to mono-nucleosome length using MNase. Proteins loosely bound to chromatin elute during this step. D. The chromatin is further fractionated by washing the nuclei in buffers with increasing salt concentration. E. The DNA is isolated from nuclei to confirm digestion of the cellular genome to 150 bp fragments. F. The quality of fractionation is tested using SDS-PAGE and Western blot (WB) for control proteins (e.g., tubulin, histone H3). The grey colored supernatants (and the pellet in case of the nuclei) represent the samples used for Western blot analysis. G. Example Western blot analysis of chromatin fractionation. Tubulin is found only in the cytoplasmic fraction and is a suitable control to test the quality of nuclear isolation. Histone H3 is a component of cellular chromatin and only elutes from nuclei in buffers with high salt. HMGB1 is a highly mobile nuclear protein (Sapojnikova et al., 2005) and thus elutes during MNase digest and under lower salt conditions. Brd1 directly binds to histone tails (Sanchez et al., 2014) and elutes under high salt conditions.

Materials and Reagents

Note: Comparable reagents from different suppliers may be used for the protocol.

  1. 150 mm tissue culture dishes (Corning, Falcon®, catalog number: 353025 )
  2. 15 ml centrifuge tube (Corning, catalog number: 430790 )
  3. 5 ml pipettes (VWR, catalog number: 89130-908 )
  4. Transfer pipette (Denville Scientific, catalog number: P7222 )
  5. 30 ml glass tube (Corning, Corex®, catalog number: 1-8445-30
    Note: This product has been discontinued.
  6. 1.7 ml microcentrifuge tubes (VWR, catalog number: 87003-294 )
  7. Pipette tips  
    0.1-10 µl (Corning, catalog number: 4153 )
    1-200 µl (Corning, catalog number: 4126 )
    100-1,000 µl (Corning, catalog number: 4129 )
  8. 250 ml sterile disposable filter units with 0.2 µm PES membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 568-0020 ) (used for Buffer I and Buffer II)
  9. 60 ml syringe (BD, catalog number: 309653 ) (used for Buffer IV.80, IV.150, IV.300 and IV.600)
  10. 25 mm syringe filter (Pall, catalog number: 4612 ) (used for Buffer IV.80, IV.150, IV.300 and IV.600)
  11. A549 cells (ATCC, catalog number: CCL-185 )
  12. Ham’s F-12K cell culture media (Thermo Fisher Scientific, GibcoTM, catalog number: 21127-022 )
  13. Fetal bovine serum (FBS) (VWR, catalog number: 89510-182 )
  14. Penicillin-streptomycin (Pen/Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  15. Trypsin-EDTA (0.25%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056 )
  16. Phosphate buffered saline (PBS) (Mediatech, catalog number: 21-030-CM )
  17. Liquid nitrogen
  18. NP-40/IGEPAL® CA-630 (Sigma-Aldrich, catalog number: I8896 ) (10% stock solution in H2O)
  19. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 ) (0.1 M stock solution in isopropanol)
  20. 1,4-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10197777001 ) (1 M stock solution in HEPES buffer, pH 7.75)
  21. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001 ) (prepared as 50x stock solution in H2O according to manufacturer instructions)
  22. Micrococcal nuclease (MNase) (Sigma-Aldrich, catalog number: N3755 ) (0.2 U/µl stock solution in H2O)
  23. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 ) (0.1 mM stock solution in H2O, pH = 10)
  24. PCR purification kit (QIAGEN, catalog number: 28104 )
  25. Orange G (Sigma-Aldrich, catalog number: O3756 ) (0.35% [w/v] orange G with 30% [w/v] sucrose in H2O for 6x stock solution)
  26. 100 bp DNA ladder (New England Biolabs, catalog number: N3231 )
  27. Broad range protein ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26623 )
  28. GelRed nucleic acid gel stain (Biotum, catalog number: 41003 )
  29. LDS sample buffer (4x) (Thermo Fisher Scientific, NovexTM, catalog number: NP0007 )
  30. Sucrose (Fisher Scientific, catalog number: BP220-1 )
  31. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 ) (1 M stock solution in H2O)
  32. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 ) (5 M stock solution in H2O)
  33. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 ) (1 M stock solution in H2O)
  34. Trizma base (Sigma-Aldrich, catalog number: T1503 ) (1 M stock solution in H2O adjusted to pH 7.4 with HCl)
  35. UltraPure agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  36. Hydrochloric acid 6.0 N solution (HCl) (Fisher Scientific, catalog number: MK-H168-4 )
  37. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 ) (0.5 M stock solution in H2O)
  38. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  39. HEPES (Sigma-Aldrich, catalog number: H3375 ) (1 M stock solution in H2O adjusted to pH 7.9 with NaOH)
  40. Sodium hydroxide (NaOH) (AMRESCO, catalog number: M137 )
  41. Buffer I.A and I.B (see Recipes)
  42. Buffer II (see Recipes)
  43. Buffer III.A and III.B (see Recipes)
  44. Buffer IV.80, IV.150, IV.300 and IV.600 (see Recipes)
  45. Hypotonic buffer (see Recipes)

Equipment

Note: Equipment with similar properties may be used for the protocol, however, we recommend using a specific kind of reusable centrifuge tubes (listed in 5) to ensure high quality isolation of nuclei.

  1. CO2 incubator for cell culture (BINDER, catalog number: 9040-0082 )
  2. Benchtop centrifuge (Beckman Coulter, model: Allegra X-14R )
  3. Rotors for benchtop centrifuge (Beckman Coulter, models: SX4750 for tissue culture and FX6100 for 10,000 x g spins, or seminal rotors suitable for high speeds)
  4. Adapters for FX6100 rotor (Beckman Coulter, catalog number: 392830 )
  5. 30 ml reusable centrifuge tubes (Sigma-Aldrich, catalog number: T2793 )
  6. Tabletop centrifuge 5424 R (Eppendorf, model: 5424 R )
  7. 1 ml tissue grinder (Dounce homogenizer) with tight fitting pestle (Ace Glass Incorporated, catalog number: 8343-01 )
  8. Water bath (Fisher Scientific, model: IsotempTM Digital-Control Water Baths Model 215 , catalog number: 15-462-15Q)
  9. Tube rotator (VWR, catalog number: 10136-084 )
  10. Pipettes  
    1-10 µl (Gilson, catalog number: F144055P )
    2-20 µl (Gilson, catalog number: F144056M )
    20-200 µl (Gilson, catalog number: F144058M )
    100-1,000 µl (Gilson, catalog number: F144059M )
  11. Agarose gel electrophoresis systems (Thermo Fisher Scientific, Thermo ScientificTM, model: Owl EasyCast B1A system )
  12. Fluorescence and chemiluminescence gel imaging system (Syngene, model: G: BOX Chemi XT4 )
  13. Heat block (Fisher Scientific, model: IsotempTM Digital Dry Baths/Block Heaters , catalog number: 88-860-022)
  14. Protein electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN® Tetra Vertical Electrophoresis Cell for Mini Precast Gels , catalog number: 1658005)
  15. Western blot apparatus (Thermo Fisher Scientific, model: SureLockTM Mini-Cell Electrophoresis System )

Software

  1. ImageJ (freely available from National Institutes of Health, https://imagej.nih.gov/ij/)

Procedure

  1. Cell culture
    Note: This protocol was optimized for nuclear isolation and chromatin fractionation of roughly 4 x 107 A549 cells (for A549 cells that is approximately two 100% confluent 150 mm cell culture dishes). This number of cells was chosen for efficient nuclear isolation as described in Procedure B. This protocol can be used for other cell types but should be optimized accordingly.
    1. For each condition, grow roughly 4 x 107 A549 cells in F-12K media with 10% FBS and 1% Pen/Strep.
      Note: Time treatments such that the required cell number is reached at the time of harvest.
    2. Harvest cells using trypsin and combine into one 15 ml tube per condition.
    3. Centrifuge for 2 min, 500 x g, room temperature using benchtop centrifuge (Allegra X-14R with rotor SX4750).
    4. Wash cells once with 10 ml of PBS and centrifuge again for 2 min, 500 x g, room temperature.
    5. Aspirate as much of the supernatant as possible without disturbing the cell pellet.
    6. Flash freeze samples in liquid nitrogen.
    7. Store samples at -80 °C until ready to process.

  2. Nuclear isolation
    Note: This section describes two alternative methods for nuclear isolation. The procedure detailed in B1 yields a clean nuclear fraction but B2 is recommended for highly mobile nuclear proteins that are lost from the nuclear fraction when mild detergents are used as described in B1 (see example in Figure 2).


    Figure 2. Fractionation of nuclear protein HMGB1 can differ depending on the method of nuclear isolation. Western blot of chromatin fractionation of A549 cells using different methods of nuclear isolation. HMGB1 is lost from nuclei during nuclear isolation B1 using NP-40 and a sucrose cushion. HMGB1 is retained in the nuclear fraction during isolation of nuclei with the alternative method described in step B2.

    1. Nuclear isolation using mild detergent and sucrose cushion.
      Note: This section describes permeabilizing the cell membrane using a mild detergent (NP-40), followed by separation of the nuclei from cytoplasmic debris by sucrose cushion. The sucrose cushion consists of a layer with 1.2 M sucrose at the bottom (Buffer II) and a layer with 0.32 M sucrose on top (Buffer I, Figure 3A). The nuclei are more dense than the 1.2 M sucrose buffer and will pellet at the bottom of the tube while the less dense cytoplasmic debris will remain in the upper sucrose layer (Figure 3B).
      1. Thaw cells on ice (about 10 min until pellet is loose).
      2. In the meantime, prepare one aliquot each of Buffer I.A and I.B by addition of PMSF, DTT, and protease inhibitor cocktail immediately before use (see Recipes for final concentrations). Add NP-40 to Buffer I.B.
      3. Gently resuspend cells in 2 ml of Buffer I.A using a 5 ml pipette. Avoid the formation of bubbles.
      4. Set aside 50 μl of the cell suspension in a new tube labeled ‘cells fraction’.
      5. Add 2 ml of Buffer I.B containing NP-40 to the 2 ml of cell suspension and mix gently by inverting the tube 2-3 times.
      6. Incubate samples on ice for 10 min. Mix gently by inverting after 5 min.
      7. Pipette 8 ml of ice-cold Buffer II into 30 ml reusable centrifuge tubes (one per sample).
      8. Carefully layer cell suspension onto Buffer II (Figure 3A).
      9. If the number of tubes is even, balance the tubes with Buffer I.A before centrifugation. If the number of tubes is uneven, use an additional tube containing water as balance.
      10. Centrifuge for 20 min, 10,000 x g, 4 °C using benchtop centrifuge (Allegra X-14R with rotor FX6100) with low brakes (deceleration setting 1 for this centrifuge model, 5-10 min of deceleration for other centrifuge models) to avoid disruption of pellet.
        Note: Use the adapters listed in the Equipment section under 4 to fit the 30 ml reusable centrifuge tubes into the FX6100 rotor. The nuclei form a dense pellet at the bottom of the tube while the cytoplasmic debris remains in the upper sucrose layer, Figure 3C.
      11. Carefully remove the supernatant using a transfer pipette. Keep pellet containing the nuclei and proceed to Procedure C.


        Figure 3. Intermediate steps of nuclear isolation. A. Layers of the sucrose cushion. 1 = cells resuspended in Buffer I containing 0.32 M sucrose. 2 = Buffer II containing 1.2 M sucrose. Clear glass tube was used for better visibility of the two layers. B. Layers of sucrose cushion as seen before the centrifugation step in the 30 ml reusable tube. Layer 1 = Buffer I. Layer 2 = Buffer II. C. Layers of sucrose cushion as seen after the centrifugation step in the 30 ml reusable tube. Layer 1 = Buffer I. Layer 2 = Buffer II. D and E. Pellet of nuclei after spin at 10,000 x g indicated by red arrow. The buffers were removed before these pictures were taken.

    2. Nuclear isolation by manual disruption
      Note: Using mild detergents such as NP-40 for nuclear isolation is important to ensure that the nuclear membrane remains largely intact. In some cases, highly mobile nuclear proteins such as HMGB1 (Sapojnikova et al., 2005) may diffuse out of the nucleus upon isolation with this method. To prevent loss of these proteins from the nuclear fraction, we adapted an alternative method that relies on a hypotonic buffer and subsequent manual disruption of the cell membrane using a Dounce homogenizer. The short centrifugation times in this step minimize diffusion of these proteins out of the nuclei (Figure 2).
      1. Thaw cells on ice (about 10 min until pellet is loose).
      2. Gently resuspend cells in 1 ml of hypotonic buffer and transfer into a 1.7 ml tube using a P1000 pipette with the tip cut (or wide orifice pipette tips) to avoid disrupting the cellular membranes.
      3. Set aside 25 μl of the cell suspension in a new tube as ‘cells fraction’.
        Note: This is proportional to the 50 μl aliquot taken from 2 ml of cell suspension in step B1.
      4. Incubate on ice for 30 min.
      5. Pre-cool the Dounce homogenizer and the pestle on ice for at least 5 min.
      6. Transfer the cell suspension to cold Dounce homogenizer using a transfer pipette.
      7. Disrupt the cell membrane using 40 strokes of the tight-fitting pestle. Minimize the formation of bubbles.
      8. Transfer sample to 1.7 ml tube using a transfer pipette.
      9. Centrifuge for 5 min, 1,500 x g, 4 °C using a tabletop centrifuge.
      10. Carefully remove the supernatant using a P1000 pipette. Transfer supernatant and label as ‘cytosol fraction’ if desired. Keep pellet containing the nuclei and proceed to Procedure C.
        Note: The nuclei form a loose pellet at the bottom of the tube that can be easily disturbed. The cytoplasmic fraction in step B1 is too dilute to isolate as a result of the sucrose cushion.

  3. Micrococcal nuclease digestion
    Note: In this step the DNA is digested to the length of mono-nucleosomes (roughly 150 bp) using MNase. While partial MNase digest in combination with nuclear fractionation by salt gradient can be used to separate euchromatin from heterochromatin (Teves and Henikoff, 2012), our protocol aims to identify global association of proteins with chromatin and does not distinguish between different types of chromatin. Subsequently, it is important that the DNA is completely digested into mono-nucleosomes to break up chromatin and allow for elution of all soluble proteins in Procedure D (Figure 4). EGTA is used instead of EDTA to stop the MNase digestion as EGTA preferentially chelates Ca2+ ions that are necessary for MNase enzymatic activity but does not impact Mg2+ ions important for protein-protein interactions. Proteins loosely bound to chromatin elute during MNase digest and can be detected in the supernatant collected in this section of the protocol.


    Figure 4. Digestion of cellular DNA using MNase. Agarose gel analysis of DNA extracted from nuclei during a time course of MNase treatment. The position of DNA bands corresponding to mono-, di- and tri-nucleosomes are indicated on the left while the DNA size in base pairs (bp) is indicated on the right. Without MNase digestion (0 min), cellular DNA barely enters the agarose gel because of the large size of the DNA molecules. After 1 min of MNase digestion, DNA fragments show the characteristic banding pattern of multiples of 150 bp. After 5 min the DNA fragments correspond to one to three nucleosomes in length. After 30 min most DNA fragments are close to 150 bp, indicating the desired digestion of cellular DNA necessary for optimal chromatin fractionation. The high concentration of DNA causes the slightly lower shift of the mono-nucleosome band. The nuclei used for this time course were isolated using method B.1. No differences in MNase digestion efficiency have been observed with nuclei isolated using method B.2.

    1. Prepare and cool Buffer III.A and III.B.
    2. Add 400 μl Buffer III.A to the nuclei.
    3. Gently resuspend the nuclei using a P1000 pipette with the tip cut (or wide orifice pipette tips) to avoid disrupting the nuclear membranes.
    4. Add 5 μl of MNase (1 U) and incubate at 37 °C (in a water bath) for 30 min.
    5. Mix every 10 min by gently inverting the tube 2-3 times.
    6. After 30 min, add 25 μl of ice-cold 0.1 M EGTA to stop the digest.
    7. Set aside a 60 μl aliquot of the nuclear suspension as ‘nuclei fraction’ and for DNA isolation to confirm the sufficient digest of DNA (described in Procedure E).
    8. Centrifuge for 10 min, 400 x g, 4 °C.
    9. Transfer supernatant to fresh tube and label ‘MNase fraction’.
    10. Wash the nuclei once by resuspending them in 400 μl Buffer III.B.
    11. Centrifuge again for 10 min, 400 x g, 4 °C. Discard the supernatant.

  4. Chromatin fractionation
    Note: In this step the nuclei are further fractionated using buffers with increasing amounts of NaCl. Proteins only weakly bound to DNA are expected to be soluble under low salt conditions and will elute from nuclei in the buffers with low NaCl concentration. Proteins tightly bound to chromatin only become soluble under high salt conditions and only elute from nuclei in the buffers with high NaCl concentration.
    1. Gently resuspend the nuclei in 400 μl Buffer IV.80 using a P1000 pipette with the tip cut (or wide orifice pipette tips).
    2. Rotate at 4 °C for 30 min.
    3. Centrifuge for 10 min, 400 x g, 4 °C.
    4. Transfer supernatant to fresh tube and label ‘80 mM fraction’.
    5. Repeat steps D1-D4 with Buffer IV.150, IV.300 and IV.600 in that order. Keep the supernatants as fractions ‘150 mM’, ‘300 mM’ and ‘600 mM’, respectively.
    6. Prepare samples for Western blot immediately (see Table 1 in Procedure F) or store at -20 °C.

  5. DNA isolation and DNA gel
    Note: In this step, DNA is isolated from the samples after MNase digestion to test the length of DNA fragments. The goal is to digest the DNA down to mono-nucleosome level of around 150 bp to ensure optimal chromatin fractionation (Figure 4). It is important that orange G or another small molecular weight dye is used for loading the DNA. Commonly used DNA loading dyes such as bromophenol blue run around the same size as 150 bp of DNA and may obscure the results.
    1. Dilute 10 μl of the ‘nuclei’ fraction in 90 μl H2O for better DNA purification.
    2. Use a PCR purification kit to isolate the DNA from this sample following the kit instructions. Elute the DNA in 30 μl of H2O.
    3. Take out 5 μl (about 5 μg) of DNA and add 1 μl of 6x Orange G. Keep the rest of the DNA on ice until results are finalized or freeze at -20 °C.
    4. Run samples and a 100 bp ladder on a 2% agarose gel containing 1x GelRed at 100 V for 1 h.
    5. Visualize the DNA using gel imager and check for prominent DNA band of around 150 bp with minimal DNA laddering (Figure 4).

  6. Verifying the quality of nuclear fractionation by Western blot
    Note: The quality of the nuclear fractionation can be analyzed using SDS-PAGE combined with Western blotting (Figure 1G). Efficient nuclear isolation can be tested by probing for proteins such as tubulin that are only found in the cytoplasm. The quality of chromatin fractionation can be tested by probing for histone proteins such as histone H3 or proteins expected to bind histone tails such as bromodomain containing protein Brd1 (Sanchez et al., 2014). The interaction of the histone H3 and Brd1 with chromatin should only be disrupted under high salt conditions. In addition, proteomics can be performed to identify changes to global chromatin composition under different conditions. Further details concerning mass spectrometry of salt fractions can be found in (Avgousti et al., 2016).  
    1. Prepare samples for analysis by Western blotting according to Table 1.

      Table 1. Preparation of different fractions for Western blot
      Fraction
      Sample
      H2O
      4x LDS sample buffer with 10% DTT
      cells (from B1) 
      10 μl
      20 μl
      10 μl
      cells (from B2)
      5 μl
      25 μl
      10 μl
      nuclei 
      10 μl
      20 μl
      10 μl
      MNase 
      30 μl 
      -
      10 μl
      80 mM
      30 μl 
      -
      10 μl
      150 mM
      30 μl 
      -
      10 μl
      300 mM
      30 μl 
      -
      10 μl
      600 mM
      30 μl 
      -
      10 μl

    2. Denature samples at 95 °C (in a heat block) for 10 min.
    3. Analyze 15 μl of each sample via SDS-PAGE and Western blot. Probe for tubulin and histone H3 as controls.

Data analysis

To compare the association of a protein of interest with chromatin under different conditions, the samples should be run on the same SDS-PAGE gel and Western blot as different exposures may confound the interpretation. Expected results for various proteins are described here to aid in data analysis, though each protein of interest tested should be considered separately. The ‘cells’ fraction represents the total amount of protein in a sample for relative comparison with other fractions. A cytoplasmic protein such as tubulin should only be present in the ‘cells’ fraction (Figure 1G). Nuclear proteins should also be present in the ‘nuclei’ fraction as seen for H3 and Brd1 (Figure 1G). The band intensity for the remaining fractions represents the solubility of nuclear proteins under these conditions. H3 and Brd1 have the highest band intensity in the ‘600 mM’ fraction, signifying greater solubility under high salt conditions typical for chromatin-associated proteins. For HMGB1 the highest band intensity can be observed for the ‘MNase’ and ‘80 mM’ fraction, indicating solubility under low salt conditions, suggesting weak association with chromatin. Differences in band intensity for different fractions can be quantified using ImageJ (freely available from National Institutes of Health, https://imagej.nih.gov/ij/).
Details concerning data analysis for mass spectrometry of salt fractions can be found in (Avgousti et al., 2016).

Notes

This protocol is suitable to compare changes in the chromatin-associated proteome under different conditions. We have used this method to show changes to cellular chromatin during infection with adenovirus (Avgousti et al., 2016).

Recipes

  1. Buffer I.A and I.B
    0.32 M sucrose
    60 mM KCl
    15 mM NaCl
    5 mM MgCl2
    0.1 mM EGTA
    15 mM Tris pH 7.4
    Filtered and stored at 4 °C
    Add fresh:
    0.5 mM DTT
    0.1 mM PMSF
    1x protease inhibitor cocktail
    Add only to Buffer I.B:
    0.1% NP-40
  2. Buffer II
    1.2 M sucrose
    60 mM KCl
    15 mM NaCl
    5 mM MgCl2
    0.1 mM EGTA
    15 mM Tris pH 7.4
    Filtered and stored at 4 °C
    Add fresh:
    0.5 mM DTT
    0.1 mM PMSF
    1x protease inhibitor cocktail
  3. Buffer III.A and III.B
    10 mM Tris pH 7.4
    2 mM MgCl2
    0.1 mM PMSF
    Add only to Buffer III.A:
    5 mM CaCl2
  4. Buffer IV.80, IV.150, IV.300 and IV.600
    10 mM Tris pH 7.4
    2 mM MgCl2
    2 mM EGTA
    0.1% Triton X-100
    Add the following concentrations of NaCl to the individual buffers:
    70 mM Buffer IV.80
    140 mM Buffer IV.150
    290 mM Buffer IV.300
    590 mM Buffer IV.600
    Filtered and stored at 4 °C
    Add fresh:
    0.1 mM PMSF
  5. Hypotonic buffer
    10 mM HEPES pH 7.9
    1.5 mM MgCl2
    10 mM KCl
    0.1 mM PMSF
    0.5 mM DTT

Acknowledgments

We thank members of the Weitzman laboratory for carefully reading and revising the protocol. This work was supported by a grant from the National Institutes of Health (CA097093), the Institute for Immunology of the University of Pennsylvania, and funds from the Children’s Hospital of Philadelphia (M.D.W.). D.C.A. was supported in part by T32 CA115299 and F32 GM112414. The protocol described herein was published in Avgousti et al., 2016.

References

  1. Avgousti, D. C., Herrmann, C., Kulej, K., Pancholi, N. J., Sekulic, N., Petrescu, J., Molden, R. C., Blumenthal, D., Paris, A. J., Reyes, E. D., Ostapchuk, P., Hearing, P., Seeholzer, S. H., Worthen, G. S., Black, B. E., Garcia, B. A. and Weitzman, M. D. (2016). A core viral protein binds host nucleosomes to sequester immune danger signals. Nature 535(7610): 173-177.
  2. Chiu, Y. F., Sugden, A. U. and Sugden, B. (2013). Epstein-Barr viral productive amplification reprograms nuclear architecture, DNA replication, and histone deposition. Cell Host Microbe 14(6): 607-618.
  3. Lam, Y. W., Evans, V. C., Heesom, K. J., Lamond, A. I. and Matthews, D. A. (2010). Proteomics analysis of the nucleolus in adenovirus-infected cells. Mol Cell Proteomics 9(1): 117-130.
  4. Sanchez, R., Meslamani, J. and Zhou, M. M. (2014). The bromodomain: from epigenome reader to druggable target. Biochim Biophys Acta 1839(8): 676-685.
  5. Sapojnikova, N., Maman, J., Myers, F. A., Thorne, A. W., Vorobyev, V. I. and Crane-Robinson, C. (2005). Biochemical observation of the rapid mobility of nuclear HMGB1. Biochim Biophys Acta 1729(1): 57-63.
  6. Simpson-Holley, M., Colgrove, R. C., Nalepa, G., Harper, J. W. and Knipe, D. M. (2005). Identification and functional evaluation of cellular and viral factors involved in the alteration of nuclear architecture during herpes simplex virus 1 infection. J Virol 79(20): 12840-12851.
  7. Teves, S. S. and Henikoff, S. (2012). Salt fractionation of nucleosomes for genome-wide profiling. Methods Mol Biol 833: 421-432.

简介

核小体是细胞染色质的核心单元,由包裹在组蛋白的八聚体周围的147个碱基对(bp)的DNA组成。蛋白质如染色质重组体,转录因子和DNA修复蛋白质与染色质动态相互作用以调控DNA的接触,控制基因转录和维持基因组完整性。与染色质结合的程度响应于诸如免疫激活,氧化应激或病毒感染等应激而迅速变化,导致对染色质构象和靶基因转录的下游作用。为了阐明在不同条件下与染色质相关蛋白质组成的变化,我们调整了现有的方案来分离核,并使用盐浓度的梯度分离细胞染色质。可以通过蛋白质印迹或质谱法评估不同盐部分中特定蛋白质的存在,从而了解与染色质相关的程度。

背景 由于与DNA或组蛋白的电荷相互作用,许多染色质相关蛋白在低盐条件下是不溶的。由于盐破坏了基于电荷的蛋白质DNA和蛋白质 - 蛋白质的相互作用,染色质相关蛋白质随着NaCl浓度的增加而变得更加可溶(Teves和Henikoff,2012)。与DNA强烈结合的蛋白质预期用高盐洗脱,而松散结合的蛋白质(例如转录因子)将用低盐洗脱。我们特别关心病毒感染如何改变与细胞染色质相关的因素的组成。核复制病毒,例如腺病毒,单纯疱疹病毒和爱泼斯坦 - 巴尔病毒,可以显着改变感染期间宿主染色质的出现(Avgousti等人,2016; Lam等人, ,2010; Simpson-Holley等人,2005; Chiu等人,2013)。我们假设外观上的这些变化部分是由于宿主染色质的蛋白质组成的差异。宿主染色质的变化可以反映细胞安装的抗病毒防御或病毒的主动操纵。为了比较未感染和感染细胞中蛋白质与染色质的关系,我们开发了该方案,使用盐梯度分级细胞核(图1)。在该方案中,我们分离核,将DNA消化至单核小体长度,然后用增加浓度的盐清洗细胞核,收集每个部分进行Western印迹分析。我们最近使用该方案来阐明在腺病毒感染期间细胞染色质的变化(Avgousti等人,2016)。我们现在将此协议作为监测蛋白质与染色质在广泛扰动条件下的关联的一般方法。


图1.核分级和示例蛋白质印迹的示意图。A.每个条件制备大约4×10 7个细胞。 B.等离子体膜透化,并且通过蔗糖垫(步骤B1)或使用Dounce均化器(步骤B2)分离核。使用MNase将DNA消化成单核小体长度。在此步骤中,蛋白质松散地结合染色质洗脱。 D.通过用增加的盐浓度在缓冲液中洗涤细胞核来进一步分级染色质。 E.从细胞核中分离DNA,以确认将细胞基因组消化至150bp片段。 F.使用SDS-PAGE和蛋白质印迹(WB)测试对照蛋白质(例如微管蛋白,组蛋白H3)的分级质量。灰色上清液(以及细胞核中的沉淀)代表用于Western印迹分析的样品。 G.实施例染色质分级分离蛋白印迹分析。微管蛋白仅在细胞质部分中发现,是测试核分离质量的合适对照。组蛋白H3是细胞染色质的组成部分,只能从高盐缓冲液中的细胞核中洗脱出来。 HMGB1是高度可移动的核蛋白(Sapojnikova等人,2005),因此在MNase消化和低盐条件下洗脱。 Brd1直接与组蛋白尾部结合(Sanchez等人,2014),并在高盐条件下洗脱。

关键字:染色质, 分馏, 盐梯度, 病毒, 染色质结合, 微球菌核酸酶

材料和试剂

注意:来自不同供应商的可比较试剂可用于该方案。

  1. 150毫米组织培养皿(Corning,Falcon ®,目录号:353025)
  2. 15ml离心管(Corning,目录号:430790)
  3. 5ml移液器(VWR,目录号:89130-908)
  4. 转移移液器(Denville Scientific,目录号:P7222)
  5. 30毫升玻璃管(Corning,Corex ®,目录号:1-8445-30) 
    注意:本产品已停产。
  6. 1.7ml微量离心管(VWR,目录号:87003-294)
  7. 移液器提示
    0.1-10μl(Corning,目录号:4153)
    1-200μl(Corning,目录号:4126)
    100-1,000μl(Corning,目录号:4129)
  8. 250毫升具有0.2微米PES膜的无菌一次性过滤器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:568-0020)(用于缓冲液I和缓冲液II)
  9. 60 ml注射器(BD,目录号:309653)(用于缓冲液IV.80,IV.150,IV.300和IV.600)
  10. 25毫米注射器过滤器(Pall,目录号:4612)(用于缓冲液IV.80,IV.150,IV.300和IV.600)
  11. A549细胞(ATCC,目录号:CCL-185)
  12. Ham's F-12K细胞培养基(Thermo Fisher Scientific,Gibco TM,目录号:21127-022)
  13. 胎牛血清(FBS)(VWR,目录号:89510-182)
  14. 青霉素 - 链霉素(Pen/Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
  15. 胰蛋白酶-EDTA(0.25%)(Thermo Fisher Scientific,Gibco TM,目录号:25200-056)
  16. 磷酸盐缓冲盐水(PBS)(Mediatech,目录号:21-030-CM)
  17. 液氮
  18. NP-40/IGEPAL CA-630(Sigma-Aldrich,目录号:I8896)(H 2 O 2中的10%储备溶液)
  19. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)(0.1M异丙醇储备溶液)
  20. 1,4-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:10197777001)(1M HEPES缓冲液中的储备溶液,pH 7.75)
  21. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11697498001)(根据制造商的说明书,在H 2 O中制备为50x储备溶液)
  22. 微球菌核酸酶(MNase)(Sigma-Aldrich,目录号:N3755)(0.2U /μlH 2 O中的储备溶液)
  23. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)(0.1mM H 2 O,pH = 10)
  24. PCR纯化试剂盒(QIAGEN,目录号:28104)
  25. 橙色G(Sigma-Aldrich,目录号:O3756)(0.35%[w/v]橙G,用于6x储备溶液的H 2 O 3中的30%[w/v]蔗糖) >
  26. 100 bp DNA ladder(New England Biolabs,目录号:N3231)
  27. 广泛的蛋白质梯(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:26623)
  28. GelRed核酸凝胶染色(Biotum,目录号:41003)
  29. LDS样品缓冲液(4x)(Thermo Fisher Scientific,Novex TM,目录号:NP0007)
  30. 蔗糖(Fisher Scientific,目录号:BP220-1)
  31. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)(1M H 2 O中的1M储备溶液)
  32. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)(H 2 O 5中的5M储备溶液)
  33. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)(1M H 2 N中的储备溶液 O)
  34. Trizma碱(Sigma-Aldrich,目录号:T1503)(H 2 O的1M储备溶液用HCl调节至pH 7.4)
  35. UltraPure琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500500)
  36. 盐酸6.0N溶液(HCl)(Fisher Scientific,目录号:MK-H168-4)
  37. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)(0.5M H 2 N中的储备溶液< O)
  38. Triton X-100(Sigma-Aldrich,目录号:T8787)
  39. 将HEPES(Sigma-Aldrich,目录号:H3375)(用NaOH调节至pH 7.9的H 2 N中的1M储备溶液)
  40. 氢氧化钠(NaOH)(AMRESCO,目录号:M137)
  41. 缓冲液I.A和I.B(见配方)
  42. 缓冲液II(参见食谱)
  43. 缓冲液III.A和III.B(见配方)
  44. 缓冲液IV.80,IV.150,IV.300和IV.600(见配方)
  45. 低音缓冲(见配方)

设备

注意:具有类似性能的设备可能用于协议,但是,我们建议使用特定类型的可重复使用的离心管(列在5中),以确保高质量的细胞核隔离。

  1. CO 2细胞培养箱(BINDER,目录号:9040-0082)
  2. 台式离心机(Beckman Coulter,型号:Allegra X-14R)
  3. 用于台式离心机的转子(Beckman Coulter,型号:SX4750用于组织培养,FX6100适用于10,000 x x 旋转,或适用于高速度的精密转子)
  4. FX6100转子适配器(Beckman Coulter,目录号:392830)
  5. 30 ml可重复使用的离心管(Sigma-Aldrich,目录号:T2793)
  6. 台式离心机5424 R(Eppendorf,型号:5424 R)
  7. 具有紧密杵的1ml组织研磨机(Dounce均化器)(Ace Glass Incorporated,目录号:8343-01)
  8. 水浴(Fisher Scientific,型号:Isotemp TM 数字控制水浴215型,目录号:15-462-15Q)
  9. 管旋转器(VWR,目录号:10136-084)
  10. 移液器
    1-10μl(Gilson,目录号:F144055P)
    2-20μl(Gilson,目录号:F144056M)
    20-200μl(Gilson,目录号:F144058M)
    100-1,000μl(Gilson,目录号:F144059M)
  11. 琼脂糖凝胶电泳系统(Thermo Fisher Scientific,Thermo Scientific TM,型号:Owl EasyCast B1A系统)
  12. 荧光和化学发光凝胶成像系统(Syngene,型号:G:BOX Chemi XT4)
  13. 热块(Fisher Scientific,型号:Isotemp TM数字干浴/块加热器,目录号:88-860-022)
  14. 蛋白质电泳仪(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels,目录号:1658005)
  15. Western印迹装置(Thermo Fisher Scientific,型号:SureLock TM 小细胞电泳系统)

软件

  1. ImageJ(可从国立卫生研究院免费获得), https://imagej.nih.gov/ij/

程序

  1. 细胞培养
    注意:该方案针对大约4×10 7个A549细胞的核分离和染色质分级进行了优化(对于约2个100%汇合的150mm细胞培养皿的A549细胞)。选择此数量的细胞用于有效的核分离,如方法B所述。该方案可用于其他细胞类型,但应相应优化。
    1. 对于每个条件,在10%FBS和1%Pen/Strep的F-12K培养基中生长大约4×10 7个 A549细胞。
      注意:在收获时达到所需的细胞数量的时间处理。
    2. 收获细胞使用胰蛋白酶,并结合成一个15毫升管每个条件
    3. 使用台式离心机(带转子SX4750的Allegra X-14R)离心2分钟,500 x g,室温。
    4. 用10ml PBS洗涤细胞一次,再次离心2分钟,500×g,室温。
    5. 吸取尽可能多的上清液,而不会干扰细胞沉淀。
    6. 在液氮中闪蒸冷冻样品
    7. 将样品储存在-80°C直到准备好处理。

  2. 核隔离
    注意:本节介绍核隔离的两种替代方法。 B1中详细描述的程序产生了一个干净的核心部分,但推荐使用B1,如B1中所述使用温和洗涤剂时,从核心部分丢失的高度可移动的核蛋白质(见图2中的示例)。 >

    图2.核蛋白HMGB1的分馏可以根据核分离方法而有所不同。使用不同的核分离方法对A549细胞的染色质分级进行Western印迹。使用NP-40和蔗糖垫,核分离期间HMGB1从细胞核中丢失。使用步骤B2中描述的替代方法,HMGB1在核分离过程中保留在核部分中
    1. 使用温和的洗涤剂和蔗糖垫进行核分离 注意:本节介绍使用温和洗涤剂(NP-40)透化细胞膜,然后通过蔗糖垫将细胞核与细胞质碎片分离。蔗糖垫层由底部含有1.2M蔗糖的层(缓冲液II)和顶部具有0.32M蔗糖的层(缓冲液I,图3A)组成。细胞核比1.2 M蔗糖缓冲液更致密,并在管的底部沉淀,而较低密度的细胞质碎片将保留在上层蔗糖层中(图3B)。
      1. 在冰上解冻细胞(约10分钟,直到颗粒松散)
      2. 同时,通过在使用前立即加入PMSF,DTT和蛋白酶抑制剂混合物来制备每个缓冲液I.A和I.B的一个等分试样(见配方的最终浓度)。将NP-40加入缓冲液I.B.
      3. 使用5ml移液管将细胞轻轻重悬于2ml缓冲液I.A中。避免形成气泡。
      4. 将50μl细胞悬浮液放在标有"细胞级分"的新管中
      5. 将2ml含有NP-40的缓冲液I.B加入2ml细胞悬浮液中,轻轻倒入管中2-3次。
      6. 将样品在冰上孵育10分钟。 5分钟后轻轻混合。
      7. 将8ml冰冷的缓冲液II吸入30 ml可重复使用的离心管中(每个样品一个)。
      8. 小心地将细胞悬液悬浮于缓冲液II上(图3A)
      9. 如果管的数量是均匀的,则在离心之前将管与缓冲液I.A平衡。如果管道数量不均匀,请使用含水的额外管道作为平衡
      10. 使用带有低制动器的台式离心机(具有转子FX6100的Allegra X-14R)离心20分钟,10,000℃,4℃,该离心机型号减速设定1,其他减速5-10分钟离心机型号)以避免颗粒破裂。
        注意:使用4号设备部分列出的适配器将30 ml可重复使用的离心管装入FX6100转子。细胞核在管的底部形成致密的颗粒,而细胞质碎片保留在上层蔗糖层中,图3C。
      11. 使用移液器小心地去除上清液。保留含有核的沉淀物,并进行到程序C.


        图3.核分离的中间步骤。 A.蔗糖垫层。 1 =细胞重悬浮于含有0.32M蔗糖的缓冲液I中。 2 =含有1.2M蔗糖的缓冲液II。使用透明玻璃管更好地了解两层。 B.在30ml可重复使用的管中离心步骤之前看到的蔗糖层的层。层1 =缓冲区I.层2 =缓冲区II。 C.在30ml可重复使用的管中离心步骤后观察到蔗糖层的层。层1 =缓冲区I.层2 =缓冲区II。 D和E.以红色箭头表示的10,000×g旋转后的核粒子。在拍摄照片之前,将缓冲区删除。

    2. 通过手动中断进行核隔离
      注意:使用温和的洗涤剂如NP-40进行核分离是重要的,以确保核膜保持在很大程度上完好无损。在某些情况下,高度移动的核蛋白如HMGB1(Sapojnikova等人,2005)可以通过该方法分离而扩散出细胞核。为了防止这些蛋白质从核部分的损失,我们调整了一种替代方法,依靠低渗缓冲液和随后使用Dounce匀浆器手动破坏细胞膜。在这个步骤中的短离心时间使这些蛋白质从细胞核中的扩散最小化(图2)
      1. 在冰上解冻细胞(约10分钟,直到颗粒松散)
      2. 轻轻将细胞悬浮于1ml低渗缓冲液中,并使用P1000移液管(或宽孔吸头)将其转移到1.7 ml管中,以避免破坏细胞膜。
      3. 将新鲜管中的细胞悬浮液25μl作为"细胞分数"放在一边。
        注意:这与步骤B1中从2ml细胞悬浮液中取出的50μl等分试样成比例。
      4. 在冰上孵育30分钟。
      5. 将Dounce均化器和杵在冰上预冷至少5分钟。
      6. 使用移液器将细胞悬浮液转移至冷的Dounce匀浆器。
      7. 使用40次紧密的杵破碎细胞膜。最小化气泡的形成。
      8. 使用转移移液管将样品转移至1.7 ml管。
      9. 使用台式离心机离心5分钟,1,500 x g,4°C。
      10. 用P1000移液管小心地清除上清液。如果需要,转移上清液并将其标记为"细胞质分数"。保留含有核的沉淀物,并进行到程序C.
        注意:细胞核在管底部形成松散的颗粒,容易受到干扰。步骤B1中的细胞质部分太稀,不能由于蔗糖垫而分离出来。

  3. 微球菌核酸酶消化
    注意:在该步骤中,使用MNase将DNA消化成单核小体的长度(约150bp)。虽然部分MNase消化与盐分离的核分离可以用于将异染色质与异染色质分离(Teves和Henikoff,2012),我们的方案旨在鉴定蛋白质与染色质的全球关联,并且不区分不同类型的染色质。随后,重要的是将DNA完全消化成单核小体以分解染色质并允许在步骤D中洗脱所有可溶性蛋白质(图4)。使用EGTA代替EDTA以停止MNase消化,因为EGTA优先螯合MNase酶活性所必需的Ca 2+离子,但不影响Mg 2 + 离子对蛋白质 - 蛋白质相互作用是重要的。蛋白质在MNase消化过程中松散地结合染色质洗脱,并且可以在方案本部分收集的上清液中检测到。


    图4.使用MNase消化细胞DNA 在MNase治疗的时间过程中从细胞核提取的DNA的琼脂糖凝胶分析。对应于单,双和三核小体的DNA条带的位置在左侧表示,而碱基对(bp)的DNA大小在右侧表示。没有MNase消化(0分钟),由于DNA分子的大小,细胞DNA几乎不能进入琼脂糖凝胶。在MNase消化1分钟后,DNA片段显示150bp倍数的特征条带图。 5分钟后,DNA片段长度对应于一至三个核小体。 30分钟后,大多数DNA片段接近150bp,表明最佳染色质分级所需的细胞DNA需要消化。 DNA的高浓度导致单核小体带的稍微偏移。使用方法B.1分离用于该时间过程的核。使用方法B.2分离细胞核后,没有观察到MNase消化效率的差异
    1. 缓冲液III.A和III.B.制备和冷却
    2. 向核中加入400μlBuffer III.A。
    3. 使用带有尖端切口的P1000移液管(或大孔吸管头)轻轻重悬核,以避免破坏核膜。
    4. 加入5μl的MNase(1U),并在37℃(在水浴中)孵育30分钟。
    5. 每10分钟轻轻翻转管2-3次
    6. 30分钟后,加入25μl冰冷的0.1M EGTA以停止消化。
    7. 将核悬浮液的60μl等分试样放在一边,将其作为"核分数"进行分离,并进行DNA分离以确认DNA的足够消化(描述于方法E中)。
    8. 离心10分钟,400 x g,4°C
    9. 将上清液转移到新鲜管中,并标记'MNase级分'
    10. 通过将其重悬在400μl缓冲液III.B中来清洗细胞核一次
    11. 再离心10分钟,400 x g,4℃。丢弃上清液。

  4. 染色质分级法 注意:在该步骤中,使用具有增加量的NaCl的缓冲液进一步分级细胞核。预期只有弱结合DNA的蛋白质在低盐条件下是可溶的,并且将在低NaCl浓度的缓冲液中从细胞核中洗脱出来。与染色质紧密结合的蛋白质仅在高盐条件下才变得可溶,只能在NaCl浓度高的缓冲液中从细胞核中洗脱出来。
    1. 使用P1000移液管轻轻重悬400μlBuffer IV.80中的细胞核(或大孔吸头)。
    2. 在4℃旋转30分钟。
    3. 离心10分钟,400 x g,4°C
    4. 将上清液转移到新鲜管中,并标记'MNase级分'
    5. 通过将其重悬在400μl缓冲液III.B中来清洗细胞核一次
    6. 再离心10分钟,400 x g,4℃。丢弃上清液。

  5. 染色质分级法 注意:在该步骤中,使用具有增加量的NaCl的缓冲液进一步分级细胞核。预期只有弱结合DNA的蛋白质在低盐条件下是可溶的,并且将在低NaCl浓度的缓冲液中从细胞核中洗脱出来。与染色质紧密结合的蛋白质仅在高盐条件下才变得可溶,只能在NaCl浓度高的缓冲液中从细胞核中洗脱出来。
    1. 使用P1000移液管轻轻重悬400μlBuffer IV.80中的细胞核(或大孔吸头)。
    2. 在4℃旋转30分钟。
    3. 离心10分钟,400 x g,4°C
    4. 将上清液转移到新鲜管中,并标记'80 mM级分'
    5. 以缓冲区IV.150,IV.300和IV.600的顺序重复步骤D1-D4。分别保持上清液分数为150mM,'300mM'和'600mM'
    6. 立即准备样品进行蛋白质印迹(见程序F中的表1)或-20℃储存。

  6. DNA分离和DNA凝胶
    注意:在该步骤中,在MNase消化后从样品中分离DNA以测试DNA片段的长度。目的是将DNA消化至约150bp的单核小体水平,以确保最佳染色质分馏(图4)。重要的是使用橙G或其他小分子量染料来加载DNA。通常使用的DNA负载染料如溴酚蓝的DNA大小与150 bp的DNA大小相同,可能会掩盖结果。
    1. 稀释10μl的"核"级分在90μlH 2 O中,以获得更好的DNA纯化。
    2. 使用PCR纯化试剂盒根据试剂盒说明书从该样品中分离DNA。在30μlH 2 O中洗脱DNA。
    3. 取出5μl(约5μg)DNA,加入1μl6x Orange G.将剩余的DNA保存在冰上,直到结果完成或-20°C冻结。
    4. 在含有1ml GelRed的2%琼脂糖凝胶上在100V下运行样品和100bp梯度1小时。
    5. 使用凝胶成像仪可视化DNA,并用最小的DNA梯度检查大约150 bp的显着DNA条带(图4)。

  7. 通过蛋白质印迹验证核分级的质量 注意:可以使用SDS-PAGE结合Western印迹分析核分馏的质量(图1G)。可以通过探测仅在细胞质中发现的蛋白质如微管蛋白来测试有效的核分离。可以通过探测组蛋白蛋白如组蛋白H3或预期结合组蛋白尾的蛋白质(例如含溴结构域的蛋白质Brd1)(Sanchez等,2014)来测试染色质分级的质量。组蛋白H3和Brd1与染色质的相互作用只能在高盐条件下被破坏。此外,可以进行蛋白质组学以鉴定不同条件下全球染色质组成的变化。有关盐馏分质谱的更多细节可以在(Avgousti et al。,2016)中找到。  
    1. 根据表1,通过Western印迹制备样品进行分析。

      表1.用于Western印迹的不同级分的制备
      分数
      示例
      H 2 O
      具有10%DTT的4x LDS样品缓冲液
      单元格(来自B1) 
      10μl
      20μl
      10μl
      单元格(来自B2)
      5μl
      25μl
      10μl
      核/
      10μl
      20μl
      10μl
      MNase 
      30μl 
      -
      10μl
      80 mM
      30μl 
      -
      10μl
      150 mM
      30μl 
      -
      10μl
      300 mM
      30μl 
      -
      10μl
      600 mM
      30μl 
      -
      10μl

    2. 在95℃(加热块)中变性样品10分钟
    3. 通过SDS-PAGE和Western印迹分析每个样品15μl。探测微管蛋白和组蛋白H3作为对照

数据分析

为了比较不同条件下感兴趣的蛋白质与染色质的关联,样品应在相同的SDS-PAGE凝胶上运行,并且不同暴露的蛋白质印迹可能会混淆解释。这里描述了各种蛋白质的预期结果,以帮助进行数据分析,尽管每个测试的蛋白质都应分开考虑。 "细胞"分数表示样品中与其他级分进行相对比较的蛋白质总量。细胞质蛋白如微管蛋白应该只存在于'细胞'部分(图1G)。核蛋白也应存在于"核"部分,如H3和Brd1所见(图1G)。剩余部分的带强度表示核蛋白在这些条件下的溶解度。 H3和Brd1在'600mM'级分中具有最高的谱带强度,这表明在染色质相关蛋白质典型的高盐条件下更高的溶解度。对于HMGB1,可以观察到"MNase"和"80mM"分数的最高频带强度,表明在低盐条件下的溶解度,表明与染色质无关。不同部分的频带强度差异可以使用ImageJ(可从National Institutes of Health,免费获得, https://imagej.nih.gov/ij/)。
有关盐馏分质谱数据分析的详细信息可参见(Avgousti等人,2016)。

笔记

该协议适用于比较不同条件下染色质相关蛋白质组的变化。我们已经使用这种方法来显示腺病毒感染期间细胞染色质的变化(Avgousti等人,2016)。

食谱

  1. 缓冲区I.A和I.B
    0.32 M蔗糖
    60 mM KCl
    15 mM NaCl
    5mM MgCl 2
    0.1 mM EGTA
    15 mM Tris pH 7.4
    过滤并储存在4°C
    添加新鲜:
    0.5 mM DTT
    0.1 mM PMSF
    1x蛋白酶抑制剂鸡尾酒
    只添加到缓冲区I.B:
    0.1%NP-40
  2. 缓冲区II
    1.2 M蔗糖
    60 mM KCl
    15 mM NaCl
    5mM MgCl 2
    0.1 mM EGTA
    15 mM Tris pH 7.4
    过滤并储存在4°C
    添加新鲜:
    0.5 mM DTT
    0.1 mM PMSF
    1x蛋白酶抑制剂鸡尾酒
  3. 缓冲区III.A和III.B 10 mM Tris pH 7.4
    2mM MgCl 2
    0.1 mM PMSF
    只添加到缓冲区III.A:
    5mM CaCl 2
  4. 缓冲液IV.80,IV.150,IV.300和IV.600
    10 mM Tris pH 7.4
    2mM MgCl 2
    2 mM EGTA
    0.1%Triton X-100
    将以下浓度的NaCl加入各缓冲液中:
    70 mM Buffer IV.80
    140 mM Buffer IV.150
    290 mM Buffer IV.300
    590 mM Buffer IV.600
    过滤并储存在4°C
    添加新鲜:
    0.1 mM PMSF
  5. 低音缓冲区
    10 mM HEPES pH 7.9
    1.5mM MgCl 2
    10 mM KCl
    0.1 mM PMSF
    0.5 mM DTT

致谢

我们感谢Weitzman实验室的成员仔细阅读和修订了该协议。这项工作得到了美国国立卫生研究院(CA097093),宾夕法尼亚大学免疫学研究所和费城儿童医院(M.D.W.)资助的资助。直升机部分由T32 CA115299和F32 GM112414支持。本文描述的方案公布于Avgousti等人,2016年。

参考文献

  1. Avgousti,DC,Herrmann,C.,Kulej,K.,Pancholi,NJ,Sekulic,N.,Petrescu,J.,Molden,RC,Blumenthal,D.,Paris,AJ,Reyes,ED,Ostapchuk,听力,P.,Seeholzer,SH,Worthen,GS,Black,BE,Garcia,BA和Weitzman,MD(2016)。  核心病毒蛋白结合宿主核小体以螯合免疫危险信号。自然 535(7610):173-177。
  2. Chiu,YF,Sugden,AU和Sugden,B.(2013)。爱泼斯坦 - 巴尔病毒生产扩增重新编码核结构,DNA复制和组蛋白沉积。细胞宿主微生物 14(6):607-618。
  3. Lam,YW,Evans,VC,Heesom,KJ,Lamond,AI和Matthews,DA(2010)。腺病毒感染细胞中核仁的蛋白质组学分析。分子细胞蛋白质组学9(1):117-130。
  4. Sanchez,R.,Meslamani,J. and Zhou,MM(2014)。  溴结构域:从表观基因组读取器到药物靶标。 Biochim Biophys Acta 1839(8):676-685。
  5. Sapojnikova,N.,Maman,J.,Myers,FA,Thorne,AW,Vorobyev,VI and Crane-Robinson,C.(2005)。生物化学观察核HMGB1的快速流动性。 Biochim Biophys Acta 1729(1):57 -63。
  6. Simpson-Holley,M.,Colgrove,RC,Nalepa,G.,Harper,JW和Knipe,DM(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/16188986"target ="_ blank">涉及单纯疱疹病毒1感染期间核结构改变的细胞和病毒因子的鉴定和功能评估。 Virol 79(20):12840-12851。
  7. Teves,SS和Henikoff,S。(2012)。用于全基因组谱分析的核小体的盐分离。方法Mol Biol 833:421-432。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Herrmann, C., Avgousti, D. C. and Weitzman, M. D. (2017). Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells. Bio-protocol 7(6): e2175. DOI: 10.21769/BioProtoc.2175.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。