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DNA Slot Blot Repair Assay
DNA斑点杂交修复试验   

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Abstract

Ultraviolet (UV) irradiation induces helix distorting photolesions such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) which threaten genomic integrity if unrepaired. In mammals, nucleotide excision repair (NER) is the only pathway that removes UV-induced DNA damages. Here we describe DNA slot blot repair assay for quantitative detection of NER activity using DNA damage specific antibodies such as anti-CPD and anti-6-4PP. Briefly, genomic DNA irradiated with UV was isolated from cells, and the genomic DNA was vacuum-transferred to a nitrocellulose membrane using a Bio-Dot SF microfiltration apparatus (Bio-Rad). A monoclonal antibody that recognizes CPD or 6-4PP was applied to detect the remaining amount of photolesions in the genomic DNA. For loading control of even loading, DNA onto the membrane can be further analyzed by SYBR-gold staining.

Keywords: DNA repair(DNA修复), Nucleotide excision repair(核苷酸切除修复), UV damage(紫外线的伤害), CPD(CPD), 6-4PP(6-4PP)

Materials and Reagents

  1. QIAamp® DNA Mini Kit (QIAGEN, catalog number: 51306 )
  2. Nitrocellulose membrane (GE Healthcare, catalog number: 10600003 )
  3. Whatman filter paper 3 MM Chr (Whatman, catalog number: 3030-917 )
  4. Mouse monoclonal antibody to CPD (Kamiya, catalog number: MC-062 )
  5. Mouse monoclonal antibody to 6-4PPs (COSMO BIO, catalog number: NMDND002 )
  6. Peroxidase affinipure goat anti-mouse IgG (H+L) (Jackson Laboratories, catalog number: 115-035-003 )
  7. SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, catalog number: S-11494 )
  8. RNase A (100 mg/ml) (Bio Basic Canada Inc., catalog number: RB0473 )
  9. Ethanol (Burdick & Jackson, catalog number: RP090-1 )
  10. PBS (Bio Basic Canada Inc., catalog number: PD8117 )
  11. Sodium azide (Junsei Chemical co., catalog number: 26628-22-8 )
  12. NaOH (Bio Basic Canada Inc., catalog number: SB0617 )
  13. EDTA (USB Corporation, catalog number: 6381-92-6 )
  14. Ammonium acetate (NH4Ac) (Katayama Chemical, catalog number: 01-4400 )
  15. Acetic acid (CH3COOH) (Merck Millipore, catalog number: 64-19-7 )
  16. NaCl (Bio Basic Canada Inc., catalog number: DB0483 )
  17. Sodium citrate (Bio Basic Canada Inc., catalog number: CB0035 )
  18. HCl (Duksan PureChemicals, catalog number: 1129 )
  19. Tris base (Bio Basic Canada Inc., catalog number: TB0196 )
  20. Tween-20 (Bio Basic Canada Inc., catalog number: 9005-65-5 )
  21. Non-fat dry milk (Sigma-Aldrich, catalog number: M7409-1BTL )
  22. PierceTM ECL Plus Western Blotting Substrate (Life Technologies, catalog number: 32132 )
  23. 0.4 M NaOH + 10 mM EDTA buffer (see Recipes)
  24. 2 M ammonium acetate (see Recipes)
  25. SSC (see Recipes)
  26. TE buffer (see Recipes)
  27. PBS-T (see Recipes)
  28. Blocking buffer (see Recipes)
  29. Buffer for primary antibody dilution (see Recipes)
  30. Buffer for secondary antibody (see Recipes)

Equipment

  1. Ultraviolet irradiator (UVP, catalog number: CL-1000 )
  2. UV-C sensor (UVP, catalog number: 97-0016-01 )
  3. Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories, catalog number: 170-6542 )
  4. Vacuum pressure pump (Gardner Denver Welch Vacuum Technology Inc., catalog number: 2522C-10 )
  5. Vacuum drier (JS Research Inc., catalog number: JSVO-30T )
  6. Orbital shaker (Wisd Laboratory Instruments, catalog number: SHO-1D )
  7. NanoDrop (Beckman Coulter, catalog number: DU-730 )
  8. Heat block (Wealtec Corp., catalog number: HB-1 )
  9. Gel doc (Bio-Rad Laboratories, catalog number: 75S/01085 )
  10. Centrifuge (Hanil Science Industrial, catalog number: Smart R17 )
  11. Microcentrifuge tube (SPL, catalog number: 60015 )
  12. 12-channel multi pipette (HTL LAB Solutions, catalog number: 5127 )
  13. Paper forcep (USBECK, catalog number: 3110)
  14. X-ray film 8 x 10 inch (Fujifilm Corporation, catalog number: HR-U30 )
  15. X-ray Cassette-AFAB TYPE 8 x 10 inch (JPI, catalog number: AFAB8x10 )

Procedure

  1. Cell culture and UV irradiation
    1. Cells were allowed to grow confluent in 6-well plates.
    2. Remove the medium, wash with warming 1x PBS once.
    3. UV irradiation (10-40 J/m2 of 254 nm UV).
      Note: It is necessary to remove the plastic lid before UV irradiation.
    4. Incubate cells in culture medium for repair for the indicated times before harvest them.
      The repair time can be varied depending on the cell’s repair capability. Thus for obtaining the optimal time courses for CPD and 6-4PP repair a pioneer experiment with various time courses should be performed. The following time courses are good to be tested for uncharacterized cells.
      Note: General repair time courses for
      CPD: No UV, 0, 6, 12, 24, 48 h
      6-4PPs: No UV, 0, 1, 2, 4, 8 h.
    5. Trypsinize and collect each sample in a 1.5 ml microcentrifuge tube by centrifugation at 2,000 x g for 30 sec.
      Cell pellets can be stored at deep freezer up to 1 month for later DNA preparation.

  2. DNA preparation (using QIAamp DNA Mini Kit)
    1. Resuspend cell pellet in 200 μl of 1x PBS (room temperature).
    2. Add 4 μl of RNase A (100 mg/ml).
    3. Add 20 μl of QIAGEN proteinase K (or protease).
    4. Add 200 μl of AL Buffer and Mix by pulse-vortexing for 15 sec.
    5. Incubate at 56 °C for 10 min.
    6. Briefly centrifuge to remove drops from the inside of the lid.
    7. Add 200 μl of EtOH (100%) and Mix again by pulse-vortexing for 15 sec.
    8. Briefly centrifuge to remove drops from the inside of the lid.
    9. Apply the mixture to QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim.
    10. Close the cap, centrifuge for 1 min at 6,000 x g.
    11. Place spin column in a clean 2 ml collection tube.
    12. Add 500 μl of AW1 buffer and centrifuge 6,000 x g, 1 min.
    13. Place spin column in a clean 2 ml collection tube.
    14. Add 500 μl of AW2 buffer and centrifuge at full speed (approximately 20,000 x g) for 3 min.
    15. Place spin column in a clean 1.5 ml E-tube.
    16. Add 200 μl of AE buffer and Incubate at RT for 5 min and centrifuge 6,000 x g, 1 min.
    17. Measure DNA concentration using NanoDrop.

  3. Slot blotting (using Bio-Dot SF microfiltration apparatus)
    1. DNA preparation (300 μl total final volume per slot)
      1. Dilute DNA.
        - CPD (50 or 100 ng) → into 150 μl of 0.4 M NaOH + 10 mM EDTA buffer
        - 6-4PP (500 ng or 1 μg) →into 150 μl of dH2O (Do not include NaOH buffer)
        Note: We used 50 ng or 100 ng of the DNA in 300 μl of total volume to 0.4 M NaOH in 10 mM EDTA for CPD detection. Whereas for 6-4PP detection we used 500 μg or 1 μg of DNA and don't treat the sample with alkali (just use the water) for 6-4PPs since it destroys the 6-4PPs).
      2. Boil for 10 min at 100 °C to denature the DNA.
      3. Transfer immediately samples to ice.
      4. Add 150 μl of cold 2 M ammonium acetate (pH 7.0) into CPD samples to neutralize the DNA (while the DNA is on ice).

    2. Slot blotter
      1. Presoak 3 blotting papers (Whatman filter 3 MM paper) and 1 nitrocellulose membrane in 6x SSC for 10 min.


      2. Cleaning the blotting Manifold (rinse it in running distilled water).
      3. Place 3 presoaked blotting papers onto the top of the manifold's middle plate. And then place nitrocellulose membrane on top of the blotting papers.
      4. Put the slotted portion of the manifold onto top of the membrane.
      5. Clamp apparatus closed using the two side pieces.


        Figure 1. Bio-Dot SF microfiltration apparatus assembly (via Bio-Rad Laboratories)

      6. Screw the plastic nozzle into the side of the apparatus.
      7. Connect vacuum line to the nozzle.
      8. Aspirate excess buffer leaking from presoaked papers and membrane.
      9. Wash the membrane once with 400 μl of TE into each slot (using multi-channel pipette) and gentle vacuum. We recommend to fill buffer in every well before applying vacuum to deliver steady pressure on each well.
      10. Carefully load DNA samples into the wells (using 1,000 p pipette) with the vacuum off and the flow valve open.


        Figure 2. Schematic table of sample loading into the well of blotting Manifold

      11. Gentle vacuum to attach DNA onto the membrane.
      12. Add 400 μl of 2x SSC to each slot and gentle vacuum.
      13. Disassemble the blotting manifold and remove the nitrocellulose membrane and put the membrane between 3 M paper.
      14. Baking the membrane in the vacuum drier at 80 °C for 2 h.

  4. Immuno-blotting
    1. Soak the blotting membrane in PBS-T for 10 min with 50 ml PBS-T in the plastic box.
    2. Transfer the membrane to 30 ml of blocking buffer and shake for 30 min at room temperature.
    3. Wash the membrane for 3 times (5 min each) with PBS-T.
    4. Apply the mouse CPD or 6-4PP primary antibody diluted 1:2,000 in PBS-T with 0.02% sodium azide.
    5. Incubate the blot in a cold room with shaking for overnight (12-14 h).
    6. Wash the membrane with PBS-T 4 times for 15 min each.
    7. Incubate with secondary antibody (anti-mouse IgG HRP-conjugated) diluted 1:2,500 in blocking buffer for 1 h at room temperature on an orbital shaker.
    8. Wash the membrane with PBS-T 4 times for 15 min each.
    9. Apply ECL and expose to film.

  5. DNA staining
    1. Rinse the membrane with PBS-T.
    2. Incubate the membrane with 10 ml of PBS-T containing SYBR-gold dye diluted 1:10,000 in PBS-T for 20 min at room temperatures on an orbital shaker.
    3. Wash the membrane with PBS-T 2 times for 5 min each.
    4. Take a photo of SYBR-gold stained blot under UV transilluminator.
      Note: The DNA bound to the membrane was detected with SYBR-Gold dye and used as loading control.

Representative data



Figure 3. Representative data for slot blot repair assay. XPA is a key factor of nucleotide exision repair for UV-induced DNA damage. Knock down of XPA using siRNA affected the repair of both CPD and the (6-4) PP. Control siRNA target cyclophilin B housekeeping gene. (A and B) Reduction in XPA level proportionally affected the repair of both CPD and the (6-4) PP as measured by slot–blot assay. Genomic DNA from cells irradiated with 10 J/m2 of UV was analyzed to detect residual CPD (A) or (6-4) PP (B) during the recovery times as indicated. C. Protein levels in human cells were analyzed by immunoblotting with the indicated antibodies from cells transfected with 100 nM siRNA targeting XPA orcyclophilin B (Control, CTRL). This figure was adapted from Kang et al. (2011).

Recipes

  1. 0.4 M NaOH + 10 mM EDTA buffer (500 ml)
    NaOH 8 g
    0.5 M EDTA 10 ml
    Adjust volume to correct volume with dH2O
  2. 2 M ammonium acetate (pH 7) (500 ml)
    Ammonium acetate (NH4Ac) 77.08 g
    Mix in ~400 ml dH2O, adjust pH to 7 with acetic acid (CH3COOH), then adjust volume to 500 ml
    Stored at 4 °C
  3. SSC (pH 7)
    1. 20x SSC (pH 7) (1 L)
      NaCl 175.3 g
      Sodium citrate 88.2 g
      Mix in ~ 700 ml dH2O, adjust pH to 7 with HCl, then adjust volume to 1 L
    2. 2x SSC (1 L)
      20x SSC 100 ml
      dH2O 900 ml
    3. 6x SSC (1 L)
      20x SSC 300 ml
      dH2O 700 ml
  4. TE buffer
    1. 1 M Tris-HCL buffer (pH 7.5) 1 L
      Mix 121.1 g Tris with 800 ml dH2O
      pH to 7.5 with HCl and add dH2O to 1 L
    2. 10x TE
      1 M Tris-HCl (pH 7.5) 100 ml
      0.5 M EDTA 20 ml
      dH2O 880 ml
    3. 1x TE
      10x TE 100 ml
      dH2O 900 ml
  5. PBS-T
    PBS with 0.1% Tween-20
  6. Blocking buffer (50 ml)
    Mix 2.5 g non-fat dry milk with 50 ml 1x PBS-T
  7. Buffer for primary antibody dilution
    Primary antibody (mouse monoclonal antibody to CPD and 6-4PP) diluted 1:2,000 in PBS-T with 0.02 % sodium azide
  8. Buffer for secondary antibody
    Secondary antibody (anti-mouse IgG HRP-conjugate) diluted 1:2,500 in blocking buffer

Acknowledgments

This work was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (1420070) and National Research Foundation (NRF) of Republic of Korea (NRF-2011-0013804 and NRF-2013-R1A2A2A04008115), and Brain Busan 21 Project.

References

  1. Kang, T. H., Reardon, J. T. and Sancar, A. (2011). Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein. Nucleic Acids Res 39(8): 3176-3187.

简介

紫外线(UV)照射诱导螺旋扭曲光致损伤,例如环丁烷嘧啶二聚体(CPD)和嘧啶 - 嘧啶酮(6-4)光产物(6-4PP),如果未修复则威胁基因组完整性。 在哺乳动物中,核苷酸切除修复(NER)是去除UV诱导的DNA损伤的唯一途径。 在这里我们描述DNA狭缝印迹修复测定NER活性使用DNA损伤特异性抗体如抗CPD和抗6-4PP的定量检测。 简言之,从细胞中分离用UV照射的基因组DNA,使用Bio-Dot SF微量过滤装置(Bio-Rad)将基因组DNA真空转移到硝酸纤维素膜上。 应用识别CPD或6-4PP的单克隆抗体来检测基因组DNA中残留的光损伤量。 对于均匀负载的上样控制,可以通过SYBR金染色进一步分析DNA在膜上。

关键字:DNA修复, 核苷酸切除修复, 紫外线的伤害, CPD, 6-4PP

材料和试剂

  1. QIAamp DNA Mini Kit(QIAGEN,目录号:51306)
  2. 硝化纤维素膜(GE Healthcare,目录号:10600003)
  3. Whatman滤纸3MM Chr(Whatman,目录号:3030-917)
  4. 针对CPD的小鼠单克隆抗体(Kamiya,目录号:MC-062)
  5. 针对6-4PP的小鼠单克隆抗体(COSMO BIO,目录号:NMDND002)
  6. 过氧化物酶亲和山羊抗小鼠IgG(H + L)(Jackson Laboratories,目录号:115-035-003)
  7. SYBR Gold Nucleic Acid Gel Stain(Life Technologies,目录号:S-11494)
  8. 核糖核酸酶A(100mg/ml)(Bio Basic Canada Inc.,目录号:RB0473)
  9. 乙醇(Burdick& Jackson,目录号:RP090-1)
  10. PBS(Bio Basic Canada Inc.,目录号:PD8117)
  11. 叠氮化钠(Junsei Chemical co。,目录号:26628-22-8)
  12. NaOH(Bio Basic Canada Inc.,目录号:SB0617)
  13. EDTA(USB公司,目录号:6381-92-6)
  14. 乙酸铵(NH 4 Ac)(Katayama Chemical,目录号:01-4400)
  15. 乙酸(CH 3 COOH)(Merck Millipore,目录号:64-19-7)
  16. NaCl(Bio Basic Canada Inc.,目录号:DB0483)
  17. 柠檬酸钠(Bio Basic Canada Inc.,目录号:CB0035)
  18. HCl(Duksan PureChemicals,目录号:1129)
  19. Tris碱(Bio Basic Canada Inc.,目录号:TB0196)
  20. Tween-20(Bio Basic Canada Inc.,目录号:9005-65-5)
  21. 脱脂奶粉(Sigma-Aldrich,目录号:M7409-1BTL)
  22. Pierce TM ECL Plus Western Blotting Substrate(Life Technologies,目录号:32132)
  23. 0.4M NaOH + 10mM EDTA缓冲液(参见配方)
  24. 2 M醋酸铵(见配方)
  25. SSC(请参阅配方)
  26. TE缓冲区(参见配方)
  27. PBS-T(参见配方)
  28. 阻止缓冲区(参见配方)
  29. 初级抗体稀释缓冲液(参见配方)
  30. 第二抗体的缓冲液(参见配方)

设备

  1. 紫外线照射器(UVP,目录号:CL-1000)
  2. UV-C传感器(UVP,目录号:97-0016-01)
  3. Bio-Dot SF微量过滤装置(Bio-Rad Laboratories,目录号:170-6542)
  4. 真空压力泵(Gardner Denver Welch Vacuum Technology Inc.,目录号:2522C-10)
  5. 真空干燥机(JS Research Inc.,目录号:JSVO-30T)
  6. 轨道摇床(Wisd Laboratory Instruments,目录号:SHO-1D)
  7. NanoDrop(Beckman Coulter,目录号:DU-730)
  8. 热块(Wealtec Corp.,目录号:HB-1)
  9. Gel doc(Bio-Rad Laboratories,目录号:75S/01085)
  10. 离心机(Hanil Science Industrial,目录号:Smart R17)
  11. 微量离心管(SPL,目录号:60015)
  12. 12通道多点移液器(HTL LAB Solutions,目录号:5127)
  13. 纸张强制(USBECK,目录号:3110)
  14. X射线胶片8×10英寸(Fujifilm Corporation,目录号:HR-U30)
  15. X-ray Cassette-AFAB TYPE 8×10英寸(JPI,目录号:AFAB8x10)

程序

  1. 细胞培养和紫外线照射
    1. 使细胞在6孔板中生长汇合
    2. 取出培养基,用1×PBS温育一次,洗涤一次
    3. UV照射(254nm UV的10-40J/m 2) 注意:在紫外线照射之前需要取下塑料盖。
    4. 孵育细胞在培养基中修复指定的时间,然后收获它们 修复时间可以根据细胞的修复而变化 能力。 因此,为了获得CPD的最佳时程 6-4PP修复先进实验用各种时间课程应该 执行。 以下时间课程是很好的测试 未表征的细胞。
      注意:
      的一般修理课程 CPD:无UV,0,6,12,24,48小时
      6-4PPs:无UV,0,1,2,4,8小时。
    5. 通过在2,000×g离心30秒,胰蛋白酶消化并收集每个样品在1.5ml微量离心管中。
      细胞沉淀可以在深冷冻器中储存长达1个月,以用于随后的DNA制备。

  2. DNA制备(使用QIAamp DNA Mini Kit)
    1. 重悬细胞沉淀在200μl的1×PBS(室温)
    2. 加入4μlRNase A(100 mg/ml)
    3. 加入20μlQIAGEN蛋白酶K(或蛋白酶)
    4. 加入200微升的AL缓冲液,并通过脉冲涡旋混合15秒
    5. 在56℃孵育10分钟。
    6. 短暂离心以除去盖子内部的液滴。
    7. 加入200μlEtOH(100%),并通过脉冲涡旋15秒再次混合
    8. 短暂离心以除去盖子内部的液滴。
    9. 将混合物加入QIAamp Mini离心柱(在2ml收集管中),不要弄湿边缘
    10. 关闭盖子,以6000xg离心1分钟。
    11. 将离心柱置于干净的2ml收集管中。
    12. 加入500μlAW1缓冲液,离心6,000×g,1分钟。
    13. 将离心柱置于干净的2ml收集管中。
    14. 加入500μlAW2缓冲液,并以全速(约20,000×em g)离心3分钟。
    15. 将离心柱置于干净的1.5 ml E管中
    16. 加入200微升AE缓冲液,并在室温孵育5分钟,离心6,000×g,1分钟。
    17. 使用NanoDrop测量DNA浓度。

  3. 槽印迹(使用Bio-Dot SF微量过滤装置)
    1. DNA制备(每个槽300μl总终体积)
      1. 稀释DNA。
        - CPD(50或100ng)→加入150μl0.4M NaOH + 10mM EDTA缓冲液中 - 6-4PP(500ng或1μg)→加入到150μldH 2 O(不包括NaOH缓冲液)中
        注意:我们在总体积300μl中使用50ng或100ng的DNA 0.4M NaOH在10mM EDTA中,用于CPD检测。 而6-4PP检测 我们使用500μg或1μg的DNA,不用碱处理样品 (只使用水)6-4PPs,因为它破坏了6-4PPs)。
      2. 在100℃煮沸10分钟以使DNA变性
      3. 立即将样品转移到冰上。
      4. 向CPD样品中加入150μl冷的2M乙酸铵(pH7.0)以中和DNA(同时DNA在冰上)。

    2. 插槽吸墨纸
      1. 在6×SSC中预凝胶3吸印纸(Whatman过滤器3MM纸)和1个硝酸纤维素膜10分钟。


      2. 清洁吸液歧管(在运行的蒸馏水中冲洗)。
      3. 将3个预浸泡的吸墨纸放在歧管的顶部 中间板。 然后将硝酸纤维素膜放在上面 吸墨纸
      4. 将歧管的开槽部分放在膜的顶部。
      5. 夹具使用两个侧件封闭

        图1. Bio-Dot SF微量过滤装置组件(通过Bio-Rad实验室)

      6. 将塑料喷嘴拧入设备侧面。
      7. 将真空管连接到喷嘴。
      8. 吸出预浸泡纸和膜渗漏的过量缓冲液
      9. 用400μlTE洗涤膜一次到每个槽(使用 多通道移液管)和温和真空。 我们建议填充缓冲区   每个井在施加真空之前在每个井上提供稳定的压力 好。
      10. 小心地将DNA样品装入孔中(使用1,000 p移液管),真空关闭,流量阀打开

        图2.将样品装入印迹歧管孔中的示意图

      11. 温和的真空将DNA粘附到膜上
      12. 在每个槽中加入400μl的2x SSC并轻微真空
      13. 拆卸吸液歧管,取出硝酸纤维素膜,将膜放在3米纸之间
      14. 将膜在真空干燥器中在80℃下烘烤2小时。

  4. 免疫印迹
    1. 浸泡在PBS-T中的印迹膜10分钟,用塑料盒中的50ml PBS-T
    2. 将膜转移到30ml封闭缓冲液,并在室温下摇动30分钟
    3. 用PBS-T洗涤膜3次(每次5分钟)
    4. 应用在PBS-T中用0.02%叠氮化钠稀释1:2000的小鼠CPD或6-4PP一抗
    5. 孵育在寒冷的房间的印迹,摇动过夜(12-14小时)
    6. 用PBS-T洗涤膜4次,每次15分钟
    7. 与第二抗体(抗小鼠IgG HRP缀合的) 在室温下在封闭缓冲液中1:2,500稀释1小时 轨道振动器
    8. 用PBS-T洗涤膜4次,每次15分钟
    9. 应用ECL并暴露于胶片。

  5. DNA染色
    1. 用PBS-T冲洗膜。
    2. 孵育膜用10毫升 的含有在PBS-T中1:10000稀释的SYBR-金染料的PBS-T中20分钟 在室温下在轨道摇床上
    3. 用PBS-T洗涤膜2次,每次5分钟
    4. 在UV透照仪下拍摄SYBR金染色的斑点的照片 注意:用SYBR-Gold染料检测与膜结合的DNA,并用作上样对照。

代表数据



图3.狭缝印迹修复测定的代表性数据。 XPA是紫外线诱导的DNA损伤的核苷酸切除修复的关键因素。使用siRNA敲低XPA影响CPD和(6-4)PP的修复。对照siRNA靶向亲环蛋白B管家基因。 (A和B)XPA水平的降低成比例地影响通过狭缝印迹测定法测量的CPD和(6-4)PP的修复。分析来自用10J/m 2 UV照射的细胞的基因组DNA以在所示的恢复时间期间检测残留的CPD(A)或(6-4)PP(B)。通过用来自用靶向XPA或家族蛋白B(对照,CTRL)的100nM siRNA转染的细胞的指示抗体通过免疫印迹分析人细胞中的蛋白质水平。该数据来自Kang等人(2011)。

食谱

  1. 0.4M NaOH + 10mM EDTA缓冲液(500ml) NaOH 8g
    0.5M EDTA 10ml 用dH <2> O调整音量以校正音量
  2. 2M乙酸铵(pH 7)(500ml) 乙酸铵(NH 4 Ac)77.08g
    在〜400ml dH 2 O中混合,用乙酸(CH 3 COOH)调节pH至7,然后将体积调节至500ml
    储存在4°C
  3. SSC(pH7)
    1. 20×SSC(pH7)(1L) NaCl 175.3g
      柠檬酸钠88.2g
      在〜700ml dH 2 O中混合,用HCl调节pH至7,然后将体积调节至1L
    2. 2x SSC(1L)
      20x SSC100ml
      dH 2 <900ml
    3. 6x SSC(1L)
      20x SSC 300ml
      dH 2 O 700ml
  4. TE缓冲区
    1. 1M Tris-HCl缓冲液(pH7.5)1L
      将121.1g Tris与800ml dH 2 O混合 用HCl将pH调节至7.5,并将dH 2 O加至1L
    2. 10x TE
      1M Tris-HCl(pH7.5)100ml
      0.5M EDTA 20ml dH 2 O 880ml
    3. 1x TE
      10x TE 100 ml
      dH 2 O 900ml
  5. PBS-T
    PBS,含有0.1%Tween-20
  6. 封闭缓冲液(50ml)
    将2.5g脱脂奶粉与50ml 1x PBS-T混合
  7. 一级抗体稀释缓冲液
    在含有0.02%叠氮化钠的PBS-T中以1:2000稀释的第一抗体(CPD和6-4PP的小鼠单克隆抗体)
  8. 二抗缓冲液
    在封闭缓冲液中以1:2,500稀释的二抗(抗小鼠IgG HRP-缀合物)

致谢

这项工作得到了国家卫生和医疗部癌症控制国家R& D项目的资助。 韩国大韩民国(1420070)和韩国国家研究基金会(NRF-2011-0013804和NRF-2013-R1A2A2A04008115)和大脑釜山21项目。

参考文献

  1. Kang,T.H.,Reardon,J.T.and Sancar,A。(2011)。 通过XPA蛋白的转录和转录后控制来调节核苷酸切除修复活性。 Nucleic Acids Res 39(8):3176-3187。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Park, J. and Kang, T. (2015). DNA Slot Blot Repair Assay. Bio-protocol 5(8): e1453. DOI: 10.21769/BioProtoc.1453.
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