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Generation of antibodies specific for a protein of interest is a common method in many disciplines. This protocol details the steps in production of a polyclonal antibody in rabbits using a bacterially expressed fusion protein as an antigen. The protocol is generated based on data presented in Wirschell et al.(2013).

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Generation of Polyclonal Specific Antibodies
制备特异性多克隆抗体

免疫学 > 抗体分析 > 抗体-抗原相互作用
作者: Maureen Wirschell
Maureen WirschellAffiliation: Department of Biochemistry, University of Mississippi Medical Center, Jackson, USA
For correspondence: mwirschell@umc.edu
Bio-protocol author page: a616
 and Mary E. Porter
Mary E. PorterAffiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, USA
Bio-protocol author page: a614
Vol 3, Iss 11, 6/5/2013, 3877 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.779

[Abstract] Generation of antibodies specific for a protein of interest is a common method in many disciplines. This protocol details the steps in production of a polyclonal antibody in rabbits using a bacterially expressed fusion protein as an antigen. The protocol is generated based on data presented in Wirschell et al.(2013).
Keywords: Antibody(抗体), Dynein(动力蛋白), Cilia(纤毛), Flagella(鞭毛), Protein purification(蛋白纯化)

[Abstract]

Materials and Reagents

  1. Topo TA cloning kit with One Shot competent cells (Life Technologies, Invitrogen™, catalog number: K457501 )
  2. pCR2.1 TOPO
  3. pET28a
  4. pMal-C (New England Biolabs)
  5. BL21 (DE3) pLysS competent cells (Stratagene, catalog number: 200132 )
  6. Bacto agar (BD Biosciences, catalog number: 214530 )
  7. Bacto Peptone Peptone (BD Biosciences, catalog number: 211677 )
  8. Bacto yeast extract (BD Biosciences, catalog number: 212750 )
  9. NaCl (Thermo Fisher Scientific, catalog number: 7647-14-5 )
  10. IPTG (Promega Corporation, catalog number: PR-V3953 )
  11. Novagen BugBuster Lysis buffer (Thermo Fisher Scientific, catalog number: 50-230-9216 )
  12. Urea (Thermo Fisher Scientific, catalog number: U15-500 )
  13. Novagen His-Bind Purification kit (Thermo Fisher Scientific, catalog number: 50-230-8606 )
  14. Novagen rLysozyme (EMD Millipore, catalog number: 71110-4 )
  15. Novagen Benzonase (EMD Millipore, catalog number: 70664-3 )
  16. PMSF (Sigma-Aldrich, catalog number: 78830-5G )
  17. Aprotinin (Sigma-Aldrich, catalog number: A1153-5MG )
  18. Roche Complete Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04-693-124-001 )
  19. Takara Chaperone plasmid set (Takara Bio Company, catalog number: 3340 )
  20. Amylose resin (New England Biolabs, catalog number: E8021S )
  21. Nitrocellulose membrane (Bio-Rad Laboratories, catalog number: 162-0115 )
  22. PVDF (EMD Millipore, catalog number: IPVH00010 )
  23. I-BLOCK (Life Technologies, Invitrogen™, catalog number: T2015 )
  24. Tween-20 (Sigma-Aldrich, catalog number: P9416 )
  25. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  26. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  27. Glycine (Sigma-Aldrich, catalog number: G8898 )
  28. Tris (Sigma-Aldrich, catalog number: T1503 )
  29. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  30. AminoLink Immobilization kit (Pierce Antibodies, catalog number: 44894 )
  31. Hydrochloric acid (HCl) (Thermo Fisher Scientific, catalog number: A144-212 )
  32. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  33. Ethanol by Acros(Thermo Fisher Scientific, catalog number: 61509-0020 )
  34. Methanol (Thermo Fisher Scientific, catalog number: BP1105-4 )
  35. Na2HPO4
  36. KH2PO4
  37. KCl
  38. NaCl
  39. CNBr-activated sepharose (Sigma-Aldrich, catalog number: C9142 )
  40. 3x PBST (see Recipes)
  41. Glycine elution buffer (see Recipes)
  42. Low pH wash buffer (see Recipes)
  43. High pH wash buffer (see Recipes)
  44. Coupling buffer (see Recipes)

Equipment

  1. Nickel-chromatography column (Thermo Fisher Scientific, catalog number: 50-230-8606 )

Procedure

  1. Preparation of expression construct:
    1. cDNA sequences encoding amino acids 155-243 of Chlamydomonas DRC1 were amplified by PCR and cloned into the pCR2.1 TOPO cloning vector to generate plasmid pMW199.1.
    2. The insert from pMW199.1 was excised with EcoR1 and subcloned into the EcoR1 site of pET28a to generate plasmid pMW219.15, which was sequenced to confirm orientation. The expressed protein sequence is shown in Figure 1.


      Figure 1. The DRC-His fusion protein. The predicted size of the expressed fusion protein is ~20 kDa. In bold is the sequence encoded by the pET28A vector including the 6-His tag (underlined) followed by the sequences encoded by the EcoR1 insert from pMW199.1 containing 6 amino acids from the pCR2.1 cloning vector and DRC1 sequences (italics).

  2. Production of the His-tagged DRC1 fusion protein: 1 ng of pMW219.15 was transformed into 50 μl of BL21 (DE3) pLysS cells from Stratagene by electroporation according to manufacturer’s recommendations.
    1. Single colonies were selected and tested for expression.
      1. Cells were grown overnight in 3 ml LB cultures at 37 °C.
      2. The next day, cultures were diluted to A600 = 0.1 and grown until A600 = 0.3 - 0.4.
      3. Expression of the His-DRC1 fusion was induced with 100 mM IPTG for 2-3 h at 37 °C.
      4. Cells were lysed and both soluble and insoluble fractions were analyzed by 15% SDS-PAGE for induction of the fusion protein.
      5. The His-DRC1 fusion protein was completely insoluble and found in inclusion bodies.  
    2. Scaled-up expression:
      1. Cells were grown in 50 ml overnight LB cultures at 37 °C.
      2. The next day, cells were diluted into 1.5 L LB to an A600 = 0.1 and grown to A600 = 0.3.
      3. Expression of the His-DRC1 fusion protein was induced with 100 mM IPTG for 2 h.

  3. Lysis of bacterial cells and purification of inclusion bodies:
    1. Cells were pelleted and resuspended in 30 ml (5 ml/gram of cell pellet) BugBuster supplemented with 10 U/ml rLysozyme, 10 U/ml Benzonase, 1 mM PMSF, 2.5 U/ml Aprotinin (Alternatively, use Roche complete protease inhibitor cocktail per manufacturers instructions). Incubate for 30 min at room temperature on a platform or rotisserie shaker.
    2. Add Triton-x-100 to 0.1% and incubate 10 min.
    3. Centrifuge at 5,000 x g for 15 min at 4 °C.
    4. Save supernatant (S1) for gel analyses. Resuspend the pellet in 0.5 volumes (15 ml) of Bugbuster diluted 1:10 with water (0.1x Bugbuster). Vortex to resuspend the pellet. Centrifuge 16,000 x g for 15 min at 4 °C.
    5. Save supernatant (S2) for gel analyses. Resuspend the pellet in 15 ml of 0.1x Bugbuster. Vortex to resuspend the pellet. Centrifuge at 16,000 x g for 15 min at 4 °C.
    6. Save supernatant (S3) for gel analyses. Resuspend the pellet in 1x binding buffer + 6 M urea. Save an aliquot for gel analyses (IB).

  4. Purification of His-DRC1 using Novagen His-Bind purification kit:
    1. Add 2.0 ml of resin to a column (20 mg/2.5 ml binding capacity) and let drain.
    2. Wash resin with 3 volumes distilled water.
    3. Wash resin with 5 volumes of 1x charge buffer (50 mM NiSO4).
    4. Wash with 3 volumes of 1x binding buffer + 6 M urea.
    5. Connect column to a peristaltic pump with a flow rate set at ~5 ml/h (0.08 ml/min). Load the solubilized inclusion bodies containing the His-DRC1 fusion protein onto the column and let run through the column. Save flow through (FT) for gel analyses.
    6. Wash column with 10 column volumes of 1x binding buffer + 6 M urea.
    7. Wash column with 6 column volumes of 1x wash buffer + 6 M urea + 20 mM Imidizole.
    8. Flush out tubing briefly and reconnect to column and peristaltic pump.
    9. Elute fusion protein in 6 column volumes of 1x elution buffer + 6 M urea and collect in 10x 300 μl fractions.
    10. Fix 50 μl of each fraction for gel analyses.
    11. Run all samples (saved supernatants, purified inclusion bodies, flowthrough, washes and column fractions) on 12% acrylamide SDS-PAGE gel (Figure 2).


      Figure 2. Coomassie stained gel of DRC1-His purification. S1, S2 and S3 are the supernatants from the three washes of the insoluble cell pellet. IB is the inclusion body pellet. FT is the flow through from the Nickel column. IB is the inclusion body pellet. 1-10 are the fractions collected from the column.

  5. Immunization of rabbits for polyclonal antibody production: The antibody against DRC1 was generated using the standard protocol offered by Spring Valley Laboratory (Woodbine, MD).
    1. First, we requested pre-immune sera from 10 rabbits. These were tested on western blots for endogenous reactivity to axonemal proteins. 2 rabbits were selected that showed minimal to no reactive bands on western blots.
    2. 4.2 mg of column purified His-DRC1 fusion protein was sent to Spring Valley Laboratories to be used as antigen for immunizations. The fusion protein was in the elution buffer used to elute the fusion protein from the Nickel-chromatography column.
    3. The following protocol was followed for immunization and bleeding:
      1. Day 0: Pre-immunization test bleed
      2. Day 0: Primary immunization
      3. Day 21: Immunogen boost
      4. Day 42: Immunogen boost
      5. Day 52: Bleed
      6. Day 63: Immunogen boost
      7. Day 73: Bleed or exsanguination
        Optional: monthly protocol extension
        Day 1: Immune boost
        Day 12: bleed
        Day 27: bleed
      8. For our DRC1 antibody production, we opted to exsanguinate after the day 73 production bleed. Approximately 1 mg of total protein is used per rabbit for the immunizations.

  6. Purification of DRC1-specific antibodies: Whole serum was tested by western blot on isolated axonemes from wild-type cells and the drc1-mutant to verify specificity of the antibody produced in rabbits. The sera was used at a 1:10,000 dilution for western blots or was further affinity purified as follows:
    1. MBP-DRC1 fusion protein:
      1. An MBP-DRC1 construct was prepared by inserting the EcoR1 insert from pMW199.1 into the EcoR1-digested pMal-C vector. Isolated clones were sequence verified.
      2. The MBP-DRC1 fusion protein was expressed in the presence of chaperone proteins to increase the solubility of the fusion protein (Takara Chaperone plasmid set).
      3. Soluble MBP-DRC1 fusion protein was purified on an amylose resin according to manufacturers instructions, run on 10% SDS-PAGE at 200 V for 1 h and transferred to nitrocellulose or PVDF membrane. The region of the membrane containing the fusion protein was cut out and used to purify DRC1-specific antibodies.
    2. Blot affinity purification:
      1. The MBP-DRC1 fusion protein was run on SDS-PAGE and transferred to two sheets of PVDF membrane. The region of the membranes containing the fusion protein were cut out and used to purify DRC1-specific antibodies.
      2. The MBP-DRC1 membrane was washed in 1x PBS, and then blocked with 5 ml of 0.2% I-BLOCK, 0.1% Tween-20 in PBS for 30 minutes.
      3. The strip was incubated overnight at room temperature with a 1:1,000 dilution of DRC1 sera in the blocking solution plus 0.05% azide to specifically bind DRC1 antibodies to the membrane.
      4. The membrane was washed 3x in PBS-Tween, and the bound DRC1 antibodies were eluted in 2 ml of glycine elution buffer for 3 min on ice.
      5. The glycine eluate was collected and neutralized with 1/10th volume of 1 M Tris pH 8.1.
      6. The elution step was repeated two more times and the three elutes were combined, normal goat serum was added to 10%, and then the eluted antibody was dialyzed overnight against 1x PBS at 4 °C.
      7. The eluted, dialyzed antibody was supplemented with 0.1% azide and stored at 4 °C.
    3. Column affinity purification:
      1. The MBP-DRC1 fusion protein was purified as indicated above and peak fractions (~10 mg) dialyzed overnight at 4 °C into coupling buffer.
      2. Next day: Prepare the CNBr-activated sepharose. The binding capacity is 13-20 mg/ml. Weigh out 0.3 grams for ~10 mg of MBP-DRC1 fusion protein and rehydrate / wash it with 1 mM HCl.
      3. Wash the column with water, then coupling buffer.
      4. Bind the MBP-DRC1 fusion to the CNBr-activated sepharose column for 2.5 h at room temperature using a rabbit pump to recirculate the protein back onto the column.
      5. Wash the column with alternating low pH wash buffer and high pH wash buffer. The column was stored in 20% ethanol until ready to use for affinity purification.
      6. Wash the MBP-DRC1 column with PBS.
      7. Cap the bottom of the column and add 2 ml of DRC1 sera and 500 μl of PBS.
      8. Mix and let the resin re-settle in the column. Incubate for 1 h.
      9. Collect the flow through.
      10. Wash the column with PBS.
      11. Elute antibody with 6 ml of 0.1 M Glycine pH 2.87.
      12. Collect 900 μl fractions into Eppendorf tubes with 100 μl of 1 M Tris pH 8.0.
      13. Pool the peak fractions and dialyze overnight into PBS.
      14. Collect the dialyzed antibodies and add sodium azide as a preservative.
    4. Pre-absorption to methanol-fixed drc1-mutant cells:
      1. The pf3 mutant cells were grown in standard Chlamydomonas media (Harris, 2009)
      2. Cells were harvested, washed and resuspended in ~50 ml of 100% methanol repeatedly until the supernatant was clear and the pellet was white. The final pellet of extracted cells was split into 5 aliquots.
      3. 50-100 microliters of antisera was diluted 1:10 into PBS plus 0.1% azide and then incubated overnight at 4 °C with the first aliquot of extracted cells.
      4. The supernatant was removed, a sample saved, and then the supernatant was incubated overnight with the 2nd aliquot of extracted cells. The process was repeated three more times.
      5. The original 1:10 dilution of antisera was diluted 1:1,000, and the supernatants from each absorption step were diluted 1:100 and 1:1,000. All samples were tested on western blots of wild-type and pf3 mutant axonemes. Aliquots with minimal background, but strong signals, were combined and used at the appropriate dilution (Wirschell et al., 2013).

Recipes

Note: All buffers can be generated using the concentrated stock solutions provided with the referenced kits and by standard molarity calculations.

  1. 3x PBST
    9.6 mM Na2HPO4
    1.5 mM KH2PO4
    3.9 mM KCl
    405 mM NaCl
    0.15% Tween-20
  2. Glycine elution buffer
    0.1 M glycine-HCl
    0.5 M NaCl
    0.05% Tween (pH 2.5)
  3. Low pH wash buffer
    0.1 M sodium acetate (pH 3.85)
    0.5 M NaCl
  4. High pH wash buffer
    0.1 M Tris (pH 9)
    0.5 M NaCl
  5. Coupling buffer
    0.1 M NaHCO3 (pH 8.0)
    0.5 M NaCl

References

  1. Harris, E. (2009). Chlamydomonas in the Laboratory. The Chlamydomonas Sourcebook. E. Harris. Kidlington, Oxford, Academic Press. 1: 241-301.
  2. Wirschell, M., Olbrich, H., Werner, C., Tritschler, D., Bower, R., Sale, W. S., Loges, N. T., Pennekamp, P., Lindberg, S., Stenram, U., Carlen, B., Horak, E., Kohler, G., Nurnberg, P., Nurnberg, G., Porter, M. E. and Omran, H. (2013). The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans. Nat Genet 45(3): 262-268. 

材料和试剂

  1. 使用One Shot感受态细胞(Life Technologies,Invitrogen TM,目录号:K457501)的Topo TA克隆试剂盒
  2. pCR2.1 TOPO
  3. pET28a
  4. pMal-C(New England Biolabs)
  5. BL21(DE3)pLysS感受态细胞(Stratagene,目录号:200132)
  6. Bacto琼脂(BD Biosciences,目录号:214530)
  7. Bacto蛋白胨蛋白胨(BD Biosciences,目录号:211677)
  8. 细菌酵母提取物(BD Biosciences,目录号:212750)
  9. NaCl(Thermo Fisher Scientific,目录号:7647-14-5)
  10. IPTG(Promega Corporation,目录号:PR-V3953)
  11. Novagen BugBuster裂解缓冲液(Thermo Fisher Scientific,目录号:50-230-9216)
  12. 尿素(Thermo Fisher Scientific,目录号:U15-500)
  13. Novagen His-Bind Purification试剂盒(Thermo Fisher Scientific,目录号:50-230-8606)
  14. Novagen rLysozyme(EMD Millipore,目录号:71110-4)
  15. Novagen Benzonase(EMD Millipore,目录号:70664-3)
  16. PMSF(Sigma-Aldrich,目录号:78830-5G)
  17. 抑肽酶(Sigma-Aldrich,目录号:A1153-5MG)
  18. Roche Complete Protease inhibitor cocktail(F.Hoffmann-La Roche,目录号:04-693-124-001)
  19. Takara Chaperone质粒组(Takara Bio公司,目录号:3340)
  20. 直链淀粉树脂(New England Biolabs,目录号:E8021S)
  21. 硝化纤维素膜(Bio-Rad Laboratories,目录号:162-0115)
  22. PVDF(EMD Millipore,目录号:IPVH00010)
  23. I-BLOCK(Life Technologies,Invitrogen TM,目录号:T2015)
  24. Tween-20(Sigma-Aldrich,目录号:P9416)
  25. Triton X-100(Sigma-Aldrich,目录号:T8787)
  26. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  27. 甘氨酸(Sigma-Aldrich,目录号:G8898)
  28. Tris(Sigma-Aldrich,目录号:T1503)
  29. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  30. AminoLink Immobilization kit(Pierce Antibodies,目录号:44894)
  31. 盐酸(HCl)(Thermo Fisher Scientific,目录号:A144-212)
  32. 乙酸钠(Sigma-Aldrich,目录号:S2889)
  33. Acros的乙醇(Thermo Fisher Scientific,目录号:61509-0020)
  34. 甲醇(Thermo Fisher Scientific,目录号:BP1105-4)
  35. Na HPO 4
  36. KH 2 PO 4
  37. KCl
  38. NaCl
  39. CNBr活化的琼脂糖凝胶(Sigma-Aldrich,目录号:C9142)
  40. 3x PBST(参见配方)
  41. 甘氨酸洗脱缓冲液(参见配方)
  42. 低pH洗涤缓冲液(见配方)
  43. 高pH洗涤缓冲液(见配方)
  44. 耦合缓冲区(参见配方)

设备

  1. 镍色谱柱(Thermo Fisher Scientific,目录号:50-230-8606)

程序

  1. 表达构建体的制备:
    1. 通过PCR扩增编码衣藻DRC1的氨基酸155-243的cDNA序列,并克隆到pCR2.1 TOPO克隆载体中以产生质粒pMW199.1。
    2. 用EcoR I切割来自pMW199.1的插入片段,并亚克隆到pET28a的Eco R 1位点以产生质粒pMW219.15,对其进行测序以确认方向。表达的蛋白质序列如图1所示

      图1.DRC-His融合蛋白。 表达的融合蛋白的预测大小约为20 kDa。粗体是由包含6-His标签(下划线)的pET28A载体编码的序列,随后是来自pMW199.1的EcoR1插入片段编码的含有来自pCR2.1克隆载体的6个氨基酸的序列和DRC1序列(斜体)。

  2. His标记的DRC1融合蛋白的产生:根据制造商的建议,通过电穿孔将1ng pMW219.15转化到来自Stratagene的50μlBL21(DE3)pLysS细胞中。
    1. 选择单个菌落并测试表达。
      1. 细胞在37℃下在3ml LB培养物中生长过夜。
      2. 第二天,将培养物稀释至A 600 = 0.1,并生长至A 600 = 0.3-0.4。
      3. 用100mM IPTG在37℃下诱导His-DRC1融合体的表达2-3小时。
      4. 裂解细胞,通过15%SDS-PAGE分析可溶性和不溶性级分以诱导融合蛋白
      5. His-DRC1融合蛋白是完全不溶的,并且在包涵体中发现。  
    2. 缩放表达式:
      1. 细胞在37℃下在50ml过夜的LB培养物中生长
      2. 第二天,将细胞稀释到1.5L LB中至A 600 = 0.1,并生长至A 600 = 0.3。
      3. 用100mM IPTG诱导His-DRC1融合蛋白的表达2小时
  3. 细菌细胞裂解和包涵体纯化:
    1. 将细胞沉淀并重悬于补充有10U/ml溶菌酶,10U/ml
    2. 加入Triton-x-100至0.1%,孵育10分钟
    3. 在4℃下以5,000xg离心15分钟。
    4. 保存上清液(S1)用于凝胶分析。用0.5倍体积(15ml)的用水(0.1×Bugbuster)1:10稀释的Bugbuster重悬沉淀。涡旋以重悬沉淀。在4℃下离心16,000xg 15分钟。
    5. 保存上清液(S2)用于凝胶分析。将沉淀重悬于15ml 0.1x Bugbuster中。涡旋以重悬沉淀。在4℃下以16,000xg离心15分钟。
    6. 保存上清液(S3)用于凝胶分析。将沉淀重悬在1×结合缓冲液+ 6M尿素中。保存一份用于凝胶分析(IB)。

  4. 使用Novagen His-Bind纯化试剂盒纯化His-DRC1:
    1. 向柱中加入2.0ml树脂(20mg/2.5ml结合容量)并排出
    2. 用3倍量的蒸馏水洗涤树脂。
    3. 用5体积的1x电荷缓冲液(50mM NiSO 4)洗涤树脂
    4. 用3倍体积的1x结合缓冲液+ 6M尿素洗涤
    5. 将柱连接至蠕动泵,流速设定为〜5ml/h(0.08ml/min)。 将含有His-DRC1融合蛋白的溶解的包涵体装载到柱上,并让其通过柱。 保存流过(FT)进行凝胶分析
    6. 用10倍柱体积的1x结合缓冲液+ 6M尿素洗涤柱
    7. 用6柱体积的1x洗涤缓冲液+ 6M尿素+ 20mM咪唑洗涤柱。
    8. 短暂排出管路,并重新连接到色谱柱和蠕动泵。
    9. 在6柱体积的1x洗脱缓冲液+ 6M尿素中洗脱融合蛋白,并收集在10×300μl级分中。
    10. 固定50微升的每个部分进行凝胶分析
    11. 在12%丙烯酰胺SDS-PAGE凝胶(图2)上运行所有样品(保存的上清液,纯化的包涵体,流过液,洗涤液和柱级分)。


      图2. DRC1-His纯化的考马斯染色的凝胶。S1,S2和S3是来自不溶性细胞沉淀的三次洗涤的上清液。 IB是包涵体沉淀。 FT是从镍柱流出的流量。 IB是包涵体沉淀。 1-10是从柱收集的级分
  5. 用于多克隆抗体生产的兔免疫:使用由Spring Valley Laboratory(Woodbine,MD)提供的标准方案产生针对DRC1的抗体。
    1. 首先,我们要求从10只兔子获得免疫前血清。这些在蛋白质印迹上测试对轴突蛋白的内源反应性。选择2只兔子,其在western印迹上显示最小至无反应性条带
    2. 将4.2mg柱纯化的His-DRC1融合蛋白送至Spring Valley Laboratories,用作免疫抗原。融合蛋白在用于洗脱融合物的洗脱缓冲液中 蛋白质。
    3. 遵循以下方案免疫和出血:
      1. 第0天:免疫前测试出血
      2. 第0天:初次免疫
      3. 第21天:免疫原促进
      4. 第42天:免疫原促进
      5. 第52天:出血
      6. 第63天:免疫原促进
      7. 第73天:出血或出血
        可选:每月协议扩展
        第1天:免疫增强
        第12天:出血
        第27天:出血
      8. 对于我们的DRC1抗体生产,我们选择在73天生产出血后放血。 每只兔子使用约1mg总蛋白用于免疫。

  6. DRC1特异性抗体的纯化:通过western印迹在来自野生型细胞和drc1 - 突变体的分离的轴突上测试全血清以验证兔中产生的抗体的特异性。血清以1:10,000稀释度用于western印迹,或如下进一步亲和纯化:
    1. MBP-DRC1融合蛋白:
      1. 通过将来自pMW199.1的EcoR1插入片段插入经EcoRI消化的pMal-C载体中制备MBP-DRC1构建体。序列验证了分离的克隆
      2. 在分子伴侣蛋白存在下表达MBP-DRC1融合蛋白以增加融合蛋白(Takara Chaperone质粒集)的溶解度。
      3. 根据制造商的说明在直链淀粉树脂上纯化可溶性MBP-DRC1融合蛋白,在200V下在10%SDS-PAGE上运行1小时,并转移至硝酸纤维素或PVDF膜。切下含有融合蛋白的膜的区域,并用于纯化DRC1特异性抗体
    2. 印迹亲和纯化:
      1. MBP-DRC1融合蛋白在SDS-PAGE上运行并转移到两片PVDF膜上。 切下含有融合蛋白的膜的区域,并用于纯化DRC1特异性抗体
      2. 将MBP-DRC1膜在1×PBS中洗涤,然后用5ml在PBS中的0.2%I-BLOCK,0.1%Tween-20封闭30分钟。
      3. 将条带在封闭溶液中加入0.05%叠氮化物的1:1000稀释的DRC1血清在室温下孵育过夜,以特异性结合膜上的DRC1抗体。
      4. 将膜在PBS-Tween中洗涤3次,结合的DRC1抗体在冰上用2ml甘氨酸洗脱缓冲液洗脱3分钟。
      5. 收集甘氨酸洗脱液,并用1/10体积的1M Tris pH 8.1中和。
      6. 将洗脱步骤再重复两次,将三次洗脱合并,将正常山羊血清加入到10%中,然后将洗脱的抗体在4℃下用1xPBS透析过夜。
      7. 洗脱,透析的抗体补充0.1%叠氮化物并在4℃下贮存
    3. 柱亲和纯化:
      1. 如上所述纯化MBP-DRC1融合蛋白,并将峰级分(〜10mg)在4℃透析过夜到偶联缓冲液中。
      2. 第二天:准备CNBr活化的琼脂糖凝胶。 结合容量为13-20mg/ml。 对于〜10mg的MBP-DRC1融合蛋白称重0.3g,并用1mM HCl再水化/洗涤。
      3. 用水洗涤柱,然后用偶联缓冲液洗涤
      4. 使用兔子泵将MBP-DRC1融合物与CNBr活化的琼脂糖凝胶柱在室温下结合2.5小时,以将蛋白质再循环回到柱上。
      5. 用交替的低pH洗涤缓冲液和高pH洗涤缓冲液洗涤柱。 将柱保存在20%乙醇中,直到准备用于亲和纯化
      6. 用PBS洗涤MBP-DRC1柱。
      7. 盖上柱的底部,并加入2ml DRC1血清和500μlPBS
      8. 混合并让树脂在柱中重新沉降。 孵育1小时。
      9. 收集流过。
      10. 用PBS清洗色谱柱。
      11. 用6ml 0.1M甘氨酸pH 2.87洗脱抗体。
      12. 收集900微升馏分到Eppendorf管用100微升的1M Tris pH 8.0
      13. 将峰组分汇集并在PBS中透析过夜。
      14. 收集透析的抗体,加入叠氮化钠作为防腐剂
    4. 预吸收至甲醇固定的drc1 - 突变细胞:
      1. 将pf3 突变体细胞在标准沙眼衣原体培养基(Harris,2009)中生长
      2. 收获细胞,洗涤并重复悬浮于〜50ml的100%甲醇中,直到上清液澄清,沉淀为白色。将提取的细胞的最终沉淀分成5等份
      3. 将50-100微升抗血清以1:10稀释到PBS加0.1%叠氮化物中,然后在4℃下用提取的细胞的第一等分试样孵育过夜。
      4. 除去上清液,保存样品,然后将上清液与提取的细胞的第二等分试样温育过夜。该过程重复三次以上。
      5. 将抗血清的原1:10稀释液以1:1000稀释,将来自每个吸收步骤的上清液以1:100和1:1000稀释。所有样品在野生型和pf3突变轴突的蛋白质印迹上进行测试。合并具有最小背景但具有强信号的等分试样,并以适当的稀释度使用(Wirschell等人,2013)。

食谱

注意: 所有缓冲液可以使用参考试剂盒提供的浓缩储备溶液和标准摩尔浓度计算生成。

  1. 3x PBST
    9.6mM Na 2 HPO 4
    1.5mM KH 2 PO 4 4/v/v 3.9 mM KCl
    405 mM NaCl 0.15%Tween-20
  2. 甘氨酸洗脱缓冲液
    0.1M甘氨酸 - 盐酸盐 0.5 M NaCl
    0.05%吐温(pH2.5)
  3. 低pH洗涤缓冲液
    0.1M乙酸钠(pH 3.85)
    0.5 M NaCl
  4. 高pH洗涤缓冲液
    0.1 M Tris(pH 9)
    0.5 M NaCl
  5. 耦合缓冲区
    0.1M NaHCO 3(pH 8.0)
    0.5 M NaCl

参考文献

  1. Harris,E。(2009)。 衣藻在实验室。 衣原体。 哈里斯。 Kidlington,Oxford,Academic Press。 1:241-301。
  2. Wearschell,M.,Olbrich,H.,Werner,C.,Tritschler,D.,Bower,R.,Sale,WS,Loges,NT,Pennekamp,P.,Lindberg,S.,Stenram,U.,Carlen, B.,Horak,E.,Kohler,G.,Nurnberg,P.,Nurnberg,G.,Porter,MEand Omran,H。(2013)。 nexin-dynein调节复合物亚基DRC1对于藻类和人类中的运动性纤毛功能是必需的。 a> Nat Genet 45(3):262-268。 
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How to cite this protocol: Wirschell, M. and Porter, M. E. (2013). Generation of Polyclonal Specific Antibodies. Bio-protocol 3(11): e779. DOI: 10.21769/BioProtoc.779; Full Text



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