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Helicase Assays
解旋酶活性检测试验   

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Abstract

Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

Keywords: Helicase assay(解旋酶活性测定), Large T-antigen(大T抗原), MCV(MCV)

Materials and Reagents

  1. HEK293
  2. Dulbecco’s modified eagle medium high glucose (DMEM) (Life Technologies, catalog number: 11965-084 )
  3. Fetal bovine serum (FBS) (Hyclone, catalog number: SH30071.03 )
  4. DPBS without Ca2+ and Mg2+ (Life Technologies, catalog number: 14190136 )
  5. Trypsin/EDTA (Life Technologies, catalog number: 25300054 )
  6. IgG sepharose6 fast flow (GE, catalog number : 17-0969-01 )
  7. M13mp18 DNA (New England Biolab, catalog number: N4040S )
  8. DNA oligo: CCAGGGTTTTCCCAGTCACGACGTTGTAAAC
  9. DNA polymerase I klenow fragment (New England Biolab, catalog number: M0210S )
  10. 100 mM dCTP (Promega Corporation, atalog number: U1221 )
  11. 100 mM dGTP (Promega Corporation, catalog number: U1211 )
  12. 100 mM dATP (Promega Corporation, catalog number: U1201 )
  13. 100 mM ATP (Roche Diagnostics, catalog number: 11140965001 )
  14. [gamma-32P] dATP
  15. DNA loading dye
  16. Polyacrylamide gel
  17. 1 M Tris (pH 7.6) (see Recipes)
  18. 0.1 M Tris (pH 7.6) (see Recipes)
  19. 5 M NaCl stock (see Recipes)
  20. 1 M MgCl2 stock (see Recipes)
  21. 10 mM MgCl2 (see Recipes)
  22. 0.1 M PMSF (Sigma-Aldrich, catalog number: 78830 ) (see Recipes)
  23. 0.1 M DTT (Sigma-Aldrich, catalog number: 43819 ) (see Recipes)
  24. 1 mg/ml Aprotinin (Roche Diagnostics, catalog number: 10236624001 ) (see Recipes)
  25. 1 mg/ml Leupeptin (Roche Diagnostics, catalog number: 11017101001 ) (see Recipes)
  26. 1 mg/ml Pepstatin A (Roche Diagnostics, catalog number: 10253286001 ) (see Recipes)
  27. 10% NP40 stock (see Recipes)
  28. 1% Bovine serum albumin (BSA) (Sigma-Aldrich) (see Recipes)
  29. 10 mg/ml BSA (see Recipes)
  30. 10x TBE buffer (see Recipes)
  31. IPP400 buffer (see Recipes)
  32. Helicase assay buffer (see Recipes)
  33. Stop buffer (see Recipes)
  34. Acrylamide gel (see Recipes)

Equipment

  1. 20 G needle (BD, catalog number: 305175 )
  2. PowerPac basic power supply (Bio-Rad Laboratories, catalog number: 164-5050 )
  3. Mini-Protean tetra cell (Bio-Rad Laboratories, catalog number: 165-8000 )
  4. Sorvall RC 6 plus centrifuge and Sorvall SA-600 rotor (Thermo Fisher Scientific)
  5. 37 °C, 5% CO2 cell culture incubator
  6. Typhoon 9410 variable mode Imager
  7. Storage phosphor screens and cassettes (GE)
  8. Fisher Vortex Genie 2 (Thermo Fisher Scientific, catalog number: 12-812 )

Procedure

  1. Protein purification and immobilization
    Note: Before this procedure, it is suggested to pre-cool centrifuge to 4 ˚C and cool down all buffers used in this procedure on ice. Performing all steps at a cold condition is optimal.
    1. 48 h post transfection (calcium phosphate assay is recommended for HEK293 cell transfection. In our experiment, a plasmid which encodes Merkel cell polyomavirus large T-antigen fusion to an IIT tag (IgG-IgG-TEV tag) was used in either 15 cm dishes or 10 cm dishes),wash transfected cells with cold PBS, scrape and re-suspend in cold PBS, and pellet at 500 x g for 5 min, 4 °C.
    2. Wash cells once with cold PBS and re-pelleted at 500 x g for 5 min, 4 °C.
    3. Wash cell pellet with IPP400 (100 µl buffer for each 1 x 107 cells. The ratio is adjustable dependent on cell line. For HEK293, a confluent 10 cm dish contains 1 x 107 cells and a confluent 15 cm dish contains 2.5 x 107 cells).
    4. Pass cells through a 20-G needle 10 times and rotate at 4 °C for 1 h.
    5. During rotation, wash IgG Sepharose6 Fast flow beads with at least 5x resin volume of PBS once. Sit on ice until most of the resin settles down. Usually, it will take 10-20 min.
    6. Remove PBS and pre-block IgG Sepharose6 Fast flow in 1% (w/v) BSA solution in PBS for at least 30 min at 4 °C with rotation. Settle down resin on ice.
    7. Spin cell lysate from step A4 at 32,572 x g for 15 min, 4 °C to get rid of debris.
    8. Remove 1% BSA from resin in step A6, mix supernatant from step A7 with pre-blocked resin from step A6. Usually, 10-15 µl resin was used for cell lysate from each 15 cm dish in our experiment.
    9. Rotate for 2 h at 4 °C.
    10. Settle down resin on ice and gently remove supernatant with a fine tip. Add at least 5 volumes of IPP400 supplemented with NP-40 at a final concentration of 0.01% (v/v). Re-suspend resin.
    11. Repeat step A10 at least twice.
    12. Wash resin with IPP400 without NP40 twice as in step A10.
    13. Settle resin down on ice and remove supernatant.
    14. Spin down resin at 500 x g for 5 min, 4 °C. Gently remove all liquid with a fine tip.
    15. Equilibrate with 10 resin volumes of 1x helicase assay buffer. Settle resin down on ice. Use immobilized protein on resins as fresh as possible. Usually, use 10 µl beads and about 1 µg protein for each reaction (to measure how many recombinant protein you get from each dish, purify protein by IgG resin following manufacturer’s protocol and briefly quantify the purified protein). The usage of protein in each reaction is adjustable.

  2. Substrate labeling
    Note: Radioactive materials are used in this procedure. Please follow standard guidelines to safely handle radioactive materials.
    1. Dissolve synthesized oligo DNA in sterilized water to a final concentration 35 ng/µl.
    2. Prepare annealing reaction as follows:
      Reagent
      Volume
      Final conc.
      M13mp18 single strand DNA (250 ng/µl)
      4 µl

      Oligo (35 ng/µl)
      1 µl

      0.1 MTris-Cl (pH7.6)
      4.3 µl
      10 mM
      10 mM MgCl2
      4.3 µl
      1 mM
      Add H2O to final volume 43 µl.


    3. Heat to 95 °C for 15 min.
    4. Briefly spin down. Incubate at 50 °C for 1 h.
    5. Briefly spin down. Slowly cool down to room temperature.
    6. Add 5 µl NEB restriction enzyme buffer 2.
    7. Add dCTP and dGTP to a final concentration 0.1 mM and 1 µl [α-32P]-dATP (3000 Ci/mM, 1 mCi/ml)
    8. Add 1 µl DNA polymerase I Klenow fragment.
    9. Incubate at room temperature for 20 min.
    10. Add 0.5 µl 10 mM dATP. Incubate at room temperature for another 20 min.
    11. Use labeled substrate as fresh as possible. Avoid freezing and thawing.
    12. Titrate labeled substrate as following: take 1, 2, 3, 4, 5 µl substrate, add sterilized water to 9 µl and add 1 µl 10x DNA loading dye. Boil 5-10 min. Load on an 11% non-denaturing polyacrylamide gel and electropherose. Dry gel and expose for autoradiography. Adjust the usage of substrate in the helicase assay depending on labeling efficiency and storage phosphor sensitivity (10-40 ng labeled DNA was used in our experiments).

  3. Helicase activity detection
    Note: Radioactive materials are used in this procedure. Please follow standard guidelines to safely handle radioactive materials.
    1. Prepare reaction buffer as follows:
      10x helicase assay buffer 5 µl
      100 mM ATP 1 µl
      H2O 44-x µl
      Gently remove 1× helicase buffer from resins in step A15. Add reaction buffer to resin.
    2. Add x µl labeled substrate (dependent on your titration) to the reaction.
    3. Incubate at 37 °C for 30 min (during this process, keep shaking tubes on a Fisher vortexer with the speed 1.5).
    4. Add 5 µl stop buffer.
    5. Briefly vortex, shortly spin down resin at room temperature and take the supernatant. Mix with 10x DNA loading dye and 5-10 µl supernatant is loaded onto an 11% non-denaturing polyacrylamide gel. Electrophorese in 1x TBE buffer.
    6. Dry gel and subject to autoradiography.

Notes

  1. Addition of 10% of glycerol in IPP400 buffer is helpful but not necessary.
  2. Immoblizing protein on other resins may also work, but the authors have not tested other resin systems.
  3. Freezing-and-thawing labeled substrates will bring unexpected bands.
  4. If you want to get rid of free labeled nucleic acid from the oligo substrate using ethenol precipitation, DNA purification kits, etc, the quality of purified substrate should be monitored to make sure the labeled short oligo is not dissociated from the M13mp18 genome.
  5. If alernative single-strand DNA, DNA oligos or radiolabeled deoxyribonucleotides (such as [α-32P] dCTP, etc.) are used, the addition of free deoxyribonucleotides in step B7 is adjustable. Noticably, your design should make sure that your radiolabeled deoxyribonucleotides have been incorporated into your substrates before the reaction is stopped by deoxyribonucleotides deficiency (In this protocol, the reaction will be stopped by dTTP deficiency.).

Recipes

  1. 1 M Tris (pH 7.6) stock
    Mix 121.14 g of Tris base with 600 ml dH2O, pH to 7.6 with HCl
    Adjust total volume to 1 liter
    Filter sterilize (0.45 μm)
    Stored at 4 °C
    1 M Tris (pH 7.5) and 1 M Tris (pH 8.0) are made at the same way
  2. 0.1 M Tris (pH 7.6)
    Add 100 µl 1M Tris (pH 7.6) into 900 µl dH2O
    Prepare fresh
  3. 5 M NaCl stock
    Mix 292.2 g sodium chloride with 800 ml dH2O
    Adjust total volume to 1 liter
    Filter sterilize (0.45 μm)
    Stored at 4 °C
  4. 1 M MgCl2 stock
    Mix 95.2 g magnesium chloride with 800 ml dH2O
    Add dH2O to a final volume of 1 L
    Filter sterilize (0.45 μm)
    Stored at 4 °C
  5. 10 mM MgCl2
    Add 10 µl 1M MgCl2 into 990 µl dH2O
    Prepare fresh
  6. 0.1 M PMSF
    Mix 174.2 mg PMSF with 10 ml ethanol
    Stored at -20 °C
  7. 0.1 M DTT
    Dissolve 1.5 g of DTT in 8 ml of H2O
    Adjust the total volume to 10 ml
    Take 100 µl, mix with 900 µl dH2O
    Stored at -20 ˚C
  8. 1 mg/ml Aprotinin
    Dissolve 20 mg aprotinin in 20 ml H2O
    Stored at -20 °C
  9. 1 mg/ml Leupeptin
    Dissolve 20 mg Leupeptin in 20 ml H2O
    Stored at -20 °C
  10. 1 mg/ml Pepstatin A
    Dissolve 20 mg Pepstatin A in 20 ml methanol
    Store at -20 °C
  11. 10% NP40 stock
    Mix 50 ml NP40 with 400 ml dH2O
    Adjust total volume to 500 ml
    Filter sterilize (0.45 μm)
    Stored at 4 °C
  12. 1% BSA
    Mix 0.1 g BSA with 400 µl dH2O
    Adjust total volume to 10 ml
    Filter sterilize (0.45 μm)
    Stored at 4 °C no more than 1 month
  13. 10 mg/ml BSA
    Mix 100 mg BSA with 10 ml dH2O
    Filter sterilize (0.45 μm)
    Stored at -20 °C
  14. 10x TBE buffer
    108 g of Tris
    55 g of boric acid
    7.5 g of EDTA, disodium salt
    Add into 800 ml dH2O then adjust total volume to 1 L
  15. IPP400 buffer
    Stock 
    20 ml
    Final conc.
    1M Tris-Cl [pH 8.0]
    200 µl
    10 mM
    5 M NaCl
    1600 µl
    400 mM
    0.1 M PMSF
    40 µl
    0.2 mM
    1 mg/ml Aprotinin
    20 µl
    1 µg/ml
    1 mg/ml Leupeptin
    20 µl
    1 µg/ml
    1 mg/ml Pepstatin A
    20 µl
    1 µg/ml
    ddH2O to 20 ml


  16. Helicase assay buffer
    Stock
    10 ml
    10x Final conc.
    1 M Tris-Cl (pH 7.5)
    2 ml
    200 mM
    1 M MgCl2
    1 ml
    100 mM
    10 mg/ml BSA
    1 ml
    1 mg/ml
    H2O
    6 ml

    Add 5 µl 0.1 M DTT to 100 µl helicase assay buffer for a final concentration of 5 mM
  17. Stop buffer
    3.3% SDS (w/v) and 0.1 M EDTA in dH2O
  18. Acrylamide gel
    Acrylamide
    Acrylamide (30%, 29:1)
    (ml)
    10x TBE
    (ml)
    ddH2O
    (ml)
    TEMED
    (µl)
    10% APs
    (µl)
    3.5%
    0.583
    0.5
    3.917
    2.5
    25
    5.0%
    0.833
    0.5
    3.667
    2.5
    25
    8.0%
    1.333
    0.5
    3.167
    2.5
    25
    11.0%
    1.833
    0.5
    3
    2.5
    25
    15.0%
    2.50
    0.5
    2.0
    2.5
    25
    20.0%
    3.333
    0.5
    1.167
    2.5
    25

Acknowledgments

This protocol is adapted from our previous publication Li et al. (2012). We acknowledge the insightful critiques of the manuscript from members from our laboratory. This study was supported by the HIV-Associated Malignancies Pilot Project Award (National Cancer Institute), National Institutes of Health (NIH) grants R01CA148768 and R01CA142723, and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

References

  1. Li, J., Wang, X., Diaz, J., Tsang, S. H., Buck, C. B. and You, J. (2013). Merkel cell polyomavirus large T antigen disrupts host genomic integrity and inhibits cellular proliferation. J Virol 87(16): 9173-9188.
  2. Stahl, H., Dröge, P. and Knippers, R. (1986). DNA helicase activity of SV40 large tumor antigen. EMBO J 5(8): 1939.
  3. Wu, C., Roy, R. and Simmons, D. T. (2001). Role of single-stranded DNA binding activity of T antigen in simian virus 40 DNA replication. J Virol 75(6): 2839-2847.

简介

解旋酶是一类酶,其是使用来自ATP水解的能量沿着核酸磷酸二酯主链(例如DNA,RNA和DNA-RNA杂交体)定向移动并分离两条退火的核酸链的运动蛋白。 许多细胞过程,例如转录,DNA复制,重组和DNA修复涉及解旋酶活性。 在这里,我们提供了一个协议,以分析解旋酶活性在体外。 在该协议中,DNA解旋酶蛋白Merkel细胞多瘤病毒大T抗原在哺乳动物细胞系HEK293中表达并固定在IgG树脂上。 进行解旋酶测定,同时蛋白质固定在IgG树脂上。

关键字:解旋酶活性测定, 大T抗原, MCV

材料和试剂

  1. HEK293
  2. Dulbecco改良的Eagle培养基高葡萄糖(DMEM)(Life Technologies,目录号:11965-084)
  3. 胎牛血清(FBS)(Hyclone,目录号:SH30071.03)
  4. 没有Ca 2+和Mg 2+的缓冲液(Life Technologies,目录号:14190136)
  5. 胰蛋白酶/EDTA(Life Technologies,目录号:25300054)
  6. IgG sepharose6 fast flow(GE,目录号:17-0969-01)
  7. M13mp18 DNA(New England Biolab,目录号:N4040S)
  8. DNA寡核苷酸:CCAGGGTTTTCCCAGTCACGACGTTGTAAAC
  9. DNA聚合酶I klenow片段(New England Biolab,目录号:M0210S)
  10. 100mM dCTP(Promega Corporation,产品编号:U1221)
  11. 100mM dGTP(Promega Corporation,目录号:U1211)
  12. 100mM dATP(Promega Corporation,目录号:U1201)
  13. 100mM ATP(Roche Diagnostics,目录号:11140965001)
  14. [gamma- 32 P] dATP
  15. DNA装载染料
  16. 聚丙烯酰胺凝胶
  17. 1 M Tris(pH 7.6)(参见配方)
  18. 0.1 M Tris(pH 7.6)(参见配方)
  19. 5 M NaCl原液(见配方)
  20. 1 M MgCl 2 原料(见配方)
  21. 10mM MgCl 2(参见配方)
  22. 0.1M PMSF(Sigma-Aldrich,目录号:78830)(参见Recipes)
  23. 0.1M DTT(Sigma-Aldrich,目录号:43819)(参见Recipes)
  24. 1mg/ml抑酶肽(Roche Diagnostics,目录号:10236624001)(参见Recipes)
  25. 1mg/ml亮抑酶肽(Roche Diagnostics,目录号:11017101001)(参见Recipes)
  26. 1mg/ml胃酶抑素A(Roche Diagnostics,目录号:10253286001)(参见Recipes)
  27. 10%NP40原料(见配方)
  28. 1%牛血清白蛋白(BSA)(Sigma-Aldrich)(参见Recipes)
  29. 10 mg/ml BSA(见配方)
  30. 10x TBE缓冲区(参见配方)
  31. IPP400缓冲区(参见配方)
  32. 解旋酶测定缓冲液(参见配方)
  33. 停止缓冲区(参见配方)
  34. 丙烯酰胺凝胶(见配方)

设备

  1. 20G针(BD,目录号:305175)
  2. PowerPac基本电源(Bio-Rad Laboratories,目录号:164-5050)
  3. Mini-Protean四细胞(Bio-Rad Laboratories,目录号:165-8000)
  4. Sorvall RC 6 plus离心机和Sorvall SA-600转子(Thermo Fisher Scientific)
  5. 37℃,5%CO 2细胞培养箱中培养
  6. 台风9410可变模式成像仪
  7. 存储荧光屏和暗盒(GE)
  8. Fisher Vortex Genie 2(Thermo Fisher Scientific,目录号:12-812)

程序

  1. 蛋白质纯化和固定
    注意:在此程序之前,建议将离心机预冷至4˚C,并在冰上冷却本程序中使用的所有缓冲液。 在寒冷的条件下执行所有步骤是最佳的。
    1. 转染后48小时(推荐用于HEK293细胞转染的磷酸钙测定)在我们的实验中,在15cm培养皿中使用编码与IIT标签(IgG-IgG-TEV标签)融合的Merkel细胞多瘤病毒大T抗原的质粒 或10cm培养皿),用冷PBS洗涤转染的细胞,刮擦并重悬于冷PBS中,并以500×g离心5分钟,4℃沉淀。
    2. 用冷PBS洗涤细胞一次,并在500×g下再沉淀5分钟,4℃。
    3. 用IPP400洗涤细胞沉淀(每个1×10 7个细胞的100μl缓冲液,比例可根据细胞系调节)对于HEK293,汇合的10cm培养皿含有1×10 7个细胞,/sup>细胞,汇合的15cm培养皿含有2.5×10 7个细胞)
    4. 使细胞通过20-G针10次,并在4℃下旋转1小时
    5. 在旋转期间,用至少5x树脂体积的PBS洗涤IgG Sepharose6 Fast flow珠一次。坐在冰上,直到大部分树脂沉降下来。通常需要10-20分钟。
    6. 在4℃下旋转除去PBS和在1%(w/v)BSA PBS溶液中的预封闭IgG Sepharose6 Fast流动至少30分钟。在冰上放置树脂。
    7. 将步骤A4中的细胞裂解物以32,572×g离心15分钟,4℃以除去碎屑。
    8. 在步骤A6中从树脂中除去1%BSA,将来自步骤A7的上清液与来自步骤A6的预封闭的树脂混合。通常,在我们的实验中,使用10-15μl树脂用于来自每个15cm培养皿的细胞裂解物
    9. 在4℃下旋转2小时。
    10. 将树脂放在冰上,轻轻地用细尖去除上清液。 加入至少5体积的补充有0.01%(v/v)的终浓度的NP-40的IPP400。 重新悬挂树脂。
    11. 重复步骤A10至少两次。
    12. 用IPP400清洗树脂,不使用NP40,按照步骤A10进行两次。
    13. 将树脂置于冰上并除去上清液。
    14. 在500℃下旋转树脂5分钟,4℃。 用细尖轻轻取出所有液体。
    15. 平衡用10树脂体积的1x解旋酶测定缓冲液。 将树脂放在冰上。 在树脂上使用尽可能新鲜的固定化蛋白质。 通常,每个使用10微升珠和约1微克蛋白质 反应(以测量从每个培养皿获得多少重组蛋白,根据制造商的方案通过IgG树脂纯化蛋白质并简单定量纯化的蛋白质)。 在每个反应中蛋白质的使用是可调节的
  2. 基材标签
    注意:本程序中使用放射性材料。 请按照标准指南安全处理放射性物质。
    1. 将合成的寡聚DNA溶解于灭菌水中至终浓度为35ng /μl
    2. 准备退火反应如下:
      试剂

      最终浓度。
      M13mp18单链DNA(250ng /μl) 4微升

      Oligo(35 ng /μl)
      1微升

      0.1 MTris-Cl(pH7.6)
      4.3微升
      10 mM
      10mM MgCl 2/
      4.3微升
      1 mM
      向最终体积43μl中加入H O

    3. 加热至95℃15分钟
    4. 短暂旋转。 在50℃孵育1小时
    5. 短暂旋转。 慢慢冷却至室温。
    6. 加入5μlNEB限制性酶缓冲液2.
    7. 加入dCTP和dGTP至终浓度0.1mM和1μl[α-32 P] -dATP(3000Ci/mM,1mCi/ml)。
    8. 加入1μlDNA聚合酶I Klenow片段
    9. 在室温下孵育20分钟。
    10. 加入0.5μl10 mM dATP。 在室温下再温育20分钟。
    11. 使用标记的底物尽可能新鲜。 避免冻融。
    12. 滴定标记的底物如下:取1,2,3,4,5μl底物,加入灭菌水至9μl,加入1μl10x DNA加载染料。煮5-10分钟。负载在11%非变性聚丙烯酰胺凝胶和电泳。干凝胶并暴露于放射自显影。根据标记效率和存储荧光灵敏度(在我们的实验中使用10-40ng标记的DNA),在解旋酶测定中调节底物的使用。

  3. 解脂酶活性检测
    注意:本程序中使用放射性材料。请按照标准指南安全处理放射性物质。
    1. 按如下方法制备反应缓冲液:
      10x解旋酶测定缓冲液5μl
      100mM ATP1μl
      H sub 2 O 44-xμl
      在步骤A15中轻轻地从树脂中除去1×解旋酶缓冲液。向树脂中加入反应缓冲液。
    2. 在反应中加入xμl标记的底物(取决于滴定)
    3. 在37℃孵育30分钟(在此过程中,保持摇动管在Fisher涡流混合器上,速度为1.5)。
    4. 加入5μl终止缓冲液
    5. 短暂涡旋,在室温下短暂旋转树脂并取上清液。 与10x DNA加载染料混合,并将5-10μl上清液加载到11%非变性聚丙烯酰胺凝胶上。 电泳在1x TBE缓冲液中。
    6. 干凝胶,并进行放射自显影

笔记

  1. 在IPP400缓冲液中加入10%甘油是有帮助的,但不是必需的
  2. 固定化蛋白在其他树脂上也可以工作,但作者没有测试其他树脂系统
  3. 冷冻和解冻标记的底物会带来意想不到的条带
  4. 如果你想使用乙烯醇沉淀,DNA纯化试剂盒等清除寡核苷酸底物上的游离标记核酸,应监测纯化底物的质量,以确保标记的短寡核苷酸不被解离来自M13mp18基因组
  5. 如果使用同种单链DNA,DNA寡核苷酸或放射性标记的脱氧核糖核苷酸(例如[α-32 P] dCTP等),则在步骤B7可调。值得注意的是,您的设计应确保您的放射性标记的脱氧核糖核苷酸已经被掺入您的底物中,然后通过脱氧核糖核苷酸缺陷停止反应(在该方案中,反应将被dTTP缺乏所阻止)。

食谱

  1. 1 M Tris(pH 7.6)储液
    将121.14g Tris碱与600ml dH 2 O混合,用HCl调节pH至7.6, 将总体积调整为1升
    过滤灭菌(0.45μm)
    储存在4°C
    以相同的方式制备1M Tris(pH7.5)和1M Tris(pH8.0)。
  2. 0.1M Tris(pH 7.6)
    将100μl1M Tris(pH7.6)加入900μldH 2 O 2/v 准备新鲜的
  3. 5 M NaCl储液
    将292.2g氯化钠与800ml dH 2 O混合 将总体积调整为1升
    过滤灭菌(0.45μm)
    储存在4°C
  4. 1 M MgCl 2 原料
    将95.2g氯化镁与800ml dH 2 O混合 将dH 2加到最终体积为1L的
    中 过滤灭菌(0.45μm)
    储存在4°C
  5. 10mM MgCl 2/
    将10μl1M MgCl 2添加到990μldH 2 O中
    准备新鲜的
  6. 0.1 M PMSF
    将174.2mg PMSF与10ml乙醇混合 储存于-20°C
  7. 0.1 M DTT
    将1.5g DTT溶解在8ml H 2 O中 将总体积调整为10 ml
    取100μl,与900μldH 2 O混合 储存在-20˚C
  8. 1mg/ml抑肽酶
    将20mg抑肽酶溶解在20ml H 2 O中 储存于-20°C
  9. 1mg/ml亮肽素 将20mg亮抑酶肽溶解在20ml H 2 O中 储存于-20°C
  10. 1mg/ml胃酶抑素A
    将20mg胃酶抑素A溶于20ml甲醇中 储存于-20°C
  11. 10%NP40股票
    将50ml NP40与400ml dH 2 O混合 将总体积调整为500 ml
    过滤灭菌(0.45μm)
    储存在4°C
  12. 1%BSA
    将0.1g BSA与400μldH 2 O混合 将总体积调整为10 ml
    过滤灭菌(0.45μm)
    储存在4°C不超过1个月
  13. 10mg/ml BSA
    将100mg BSA与10ml dH 2 O混合 过滤灭菌(0.45μm)
    储存于-20°C
  14. 10x TBE缓冲区
    108克Tris
    55克硼酸 7.5g EDTA,二钠盐
    加入到800ml dH 2 O中,然后将总体积调节到1L
  15. IPP400缓冲液
    股票
    20ml
    最终浓度。
    1M Tris-Cl [pH 8.0]
    200μl
    10 mM
    5 M NaCl
    1600微升
    400 mM
    0.1 M PMSF
    40微升
    0.2 mM
    1mg/ml抑肽酶
    20微升
    1μg/ml
    1mg/ml亮肽素 20微升
    1μg/ml
    1mg/ml胃酶抑素A
    20微升
    1μg/ml
    ddH 2 O至20ml

  16. 解旋酶测定缓冲液
    库存
    10 ml
    10x最终浓度。
    1M Tris-Cl(pH7.5) 2 ml
    200 mM
    1 M MgCl 2
    1 ml
    100 mM
    10mg/ml BSA
    1 ml
    1 mg/ml
    H sub 2 O
    6毫升

    加入5μl0.1M DTT到100μl解旋酶测定缓冲液中,终浓度为5mM
  17. 停止缓冲区
    3.3%SDS(w/v)和0.1M EDTA在dH 2 O中
  18. 丙烯酰胺凝胶
    丙烯酰胺
    丙烯酰胺(30%,29:1)
    (ml)
    10x TBE
    (ml)
    ddH sub 2 O
    (ml)
    TEMED
    (μl)
    10%AP
    (μl)
    3.5%
    0.583
    0.5
    3.917
    2.5
    25
    5.0%
    0.833
    0.5
    3.667
    2.5
    25
    8.0%
    1.333
    0.5
    3.167
    2.5
    25
    11.0%
    1.833
    0.5
    3
    2.5
    25
    15.0%
    2.50
    0.5
    2.0
    2.5
    25
    20.0%
    3.333
    0.5
    1.167
    2.5
    25

致谢

该协议改编自我们之前的出版物Li 等人(2012)。 我们承认来自我们实验室成员的手稿的有见地的批评。 这项研究得到艾滋病相关恶性肿瘤试验项目奖(国家癌症研究所),国家卫生研究院(NIH)拨款R01CA148768和R01CA142723,以及NIH,国家癌症研究所,癌症研究中心的校内研究计划的支持。

参考文献

  1. Li,J.,Wang,X.,Diaz,J.,Tsang,S.H.,Buck,C.B.and You,J.(2013)。 默克尔细胞多瘤病毒大T抗原破坏宿主基因组完整性, 抑制细胞增殖。 Virol 87(16):9173-9188。
  2. Stahl,H.,Dröge,P。和Knippers,R。(1986)。 SV40大肿瘤抗原的DNA解旋酶活性。 EMBO J ; 5(8):1939.
  3. Wu,C.,Roy,R。和Simmons,D.T。(2001)。 T抗原的单链DNA结合活性在猿猴病毒40 DNA复制中的作用。/a> J Virol 75(6):2839-2847
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, X., Li, J., Diaz, J. and You, J. (2014). Helicase Assays. Bio-protocol 4(6): e1079. DOI: 10.21769/BioProtoc.1079.
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