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This protocol describes a method of purifying antibodies from sera with denatured antigens immobilized on western blot membranes. Advantages include (1) fast and easy; (2) purification of antibody with antigen in denatured form allows high yield in case antigen protein solubility is limited. Disadvantage is that possible antibodies that recognize certain 3D structure in solution of antigens might not be purified using such a method. Regarding this issue, the flow through is recommended to be kept and can be used for other purification methods with folded antigens.

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Antibody Purification from Western Blotting
利用免疫印迹法(WB)进行抗体纯化

免疫学 > 抗体分析 > 抗体-抗原相互作用
作者: Lin Fang
Lin FangAffiliation: Department of Pediatrics, School of Medicine, Stanford University, Stanford, USA
For correspondence: cheerfulfang@hotmail.com
Bio-protocol author page: a20
Vol 2, Iss 6, 3/20/2012, 9099 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.133

[Abstract] This protocol describes a method of purifying antibodies from sera with denatured antigens immobilized on western blot membranes. Advantages include (1) fast and easy; (2) purification of antibody with antigen in denatured form allows high yield in case antigen protein solubility is limited. Disadvantage is that possible antibodies that recognize certain 3D structure in solution of antigens might not be purified using such a method. Regarding this issue, the flow through is recommended to be kept and can be used for other purification methods with folded antigens.

[Abstract] 本指南介绍了一种从固定在免疫印迹膜上含有变性抗原的血清中纯化抗体的方法。优点包括:(1)快速且容易;(2)抗原蛋白溶解度有限的情况下从含变性抗原的血清中纯化抗体产量更高。缺点是抗体识别抗原的可能性三维结构在抗原溶液中无法用这种方法纯化出来。由于这个原因,建议保留过滤液,可以用其他纯化方法纯化出折叠抗原。

Materials and Reagents

  1. Antigen protein
  2. PVDF or Nitrocellulose membrane (Amersham biosciences/GE Healthcare Dharmacon)
  3. Ponceau S (Sigma-Aldrich, catalog number: P7170 )
  4. non-fat Milk powder (Carnation, any your favorite brand in local store)
  5. Tween 20 (Sigma-Aldrich)
  6. Glycine
  7. Acetic acid
  8. 2 M Tris-HCl (pH 8.5)
  9. NaCl
  10. KCl
  11. Na2HPO4
  12. KH2PO4
  13. Ultra pure water
  14. TBST (see Recipes)
  15. 10x TBS (1 L) (see Recipes)
  16. PBS (pH 7.4) (see Recipes)
  17. Ponceau S solution (see Recipes)

Equipment

  1. Dialysis bag (Thermo Fisher Scientific/Pierce Antibodies, catalog number: 68035 )
  2. Comb

Procedure

  1. Load 1-10 mg purified antigen protein into appropriate SDS-PAGE gel.
    Note 1: High amount of antigen protein is essential to retrieve antibody in high purity and yield. To load as much protein into the gel as possible:
    1. Use wide comb (vendors normally have comb with only two wells: one well is in regular size for marker, the other well takes the rest of the space).
    2. Insoluble protein can be purified under denaturing conditions.
    3. Even for soluble protein, higher yield can be achieved by eluting directly from the purification beads by SDS loading buffer.
    Note 2: If your protein has a big tag such as GST, MBP, NusA, you should always run a preclearance of antibody against these tags from the serum with blot containing these tag proteins.
  2. Transfer to PVDF or Nitrocellulose membrane (note, if your protein size is small, choose 0.2 μm pore size rather than 0.45 μm).
  3. Stain the membrane with Ponceau S solution and cut the strip containing your antigen protein as small as possible.
  4. Pre-elute the membrane with 100 mM glycine (pH 2.5) for 10 min. Rinse with TBST.
  5. Block with 5% non-fat milk in TBST for 1 h at room temperature (RT).
  6. Rinse with TBST three times.
  7. Incubate the 5-10 ml antiserum overnight at 4 °C. Depending on the membrane size.
  8. Wash with TBST three times.
  9. Elute the antibody by incubating the membrane with 1-2 ml 100 mM Glycine (pH 2.5) for 2 min.
  10. While incubating, add 75 μl-100 μl 2 M Tris-HCl (pH 8.5) to tubes. The volume should allow a final concentration of 150 μM Tris after step 11.
  11. After 2 min, add 1-2 ml of the eluted antibody in glycine from step 9 to the tubes from step 10. The Tris (pH 8.5) should neutralize the acidic glycine.
  12. Repeat step 9-11 for three times.
  13. Dialyze the fractions against PBS (pH 7.4) and determine antibody concentration as your regular protein work.
  14. Test the antibody with western, immunoprecipitation and immunofluorescence.

Recipes

  1. 10x TBS (1 L)
    24.23 g Tris
    80.06 g NaCl
    Mix in 800 ml water
    pH to 7.6 with HCl
    Adjust volume to 1 L.
  2. TBST (1 L)
    100 ml of 10x TBS
    900 ml ultra pure water
    500 μl Tween 20
    Note: Tween 20 is very viscous and will stick to the tip of your measuring pipettes. Be sure you take the right amount and add in buffer by pipetting up and down. At the same time, the buffer should be stirring.
  3. PBS (pH 7.4) (1 L)
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    800 ml H2O
    Adjust pH to 7.4.
    Adjust volume to 1 L with additional distilled H2O.
    Sterilize by autoclaving
  4. Ponceau S solution (1 L)
    1 g Ponceau S
    50 ml Acetic acid
    Add H2O to 1 L

Acknowledgments

This protocol was adapted from Kurien (2009) and developed in Guowei Fang’s lab, Departmental of Biological Sciences, Stanford University, CA, USA. Funding from the NIH supported this work.

References

  1. Kurien, B. T. (2009). Affinity purification of autoantibodies from an antigen strip excised from a nitrocellulose protein blot. Methods Mol Biol 536: 201-211.

材料和试剂

  1. 抗原蛋白
  2. PVDF或硝化纤维素膜(Amersham biosciences/GE Healthcare Dharmacon)
  3. Ponceau S(Sigma-Aldrich,目录号:P7170)
  4. 脱脂奶粉(康乃馨,任何您喜欢的品牌在本地商店)
  5. 吐温20(Sigma-Aldrich)
  6. 甘氨酸
  7. 乙酸
  8. 2 M Tris-HCl(pH 8.5)
  9. NaCl
  10. KCl
  11. Na HPO 4
  12. KH 2 PO 4
  13. 超纯水
  14. TBST(参见配方)
  15. 10x TBS(1升)(参见配方)
  16. PBS(pH 7.4)(参见配方)
  17. Ponceau S解决方案(参见配方)

设备

  1. 透析袋(Thermo Fisher Scientific/Pierce Antibodies,目录号:68035)
  2. 梳子

程序

  1. 将1-10mg纯化的抗原蛋白加载到合适的SDS-PAGE凝胶中 注意1:高量的抗原蛋白对于以高纯度和产率回收抗体是必需的。 尽可能加载尽可能多的蛋白质到凝胶中:
    1. 使用宽梳(供应商通常有梳子,只有两口井:一个井是常规大小的标记,另一个井占用空间的其余部分)。
    2. 不溶性蛋白质可以在变性条件下纯化
    3. 即使对于可溶性蛋白质,可以通过用SDS上样缓冲液直接从纯化珠洗脱来实现更高的产量。
    注意2:如果你的蛋白质有一个大的标签,如GST,MBP,NusA,你应该总是运行一个预防措施的抗这些标签的抗体从含有这些标签蛋白的印迹的血清。
  2. 转移到PVDF或硝化纤维素膜(注意,如果你的蛋白质尺寸小,选择0.2μm孔径而不是0.45μm)。
  3. 用Ponceau S溶液染色膜,切割含有您的抗原蛋白的条带尽可能小。
  4. 用100mM甘氨酸(pH 2.5)预洗脱膜10分钟。用TBST冲洗。
  5. 在室温(RT)下用TBST中的5%脱脂牛奶封闭1小时。
  6. 用TBST冲洗三次。
  7. 在4℃下孵育5-10ml抗血清过夜。 取决于膜的大小。
  8. 用TBST洗三次。
  9. 通过将膜与1-2ml 100mM甘氨酸(pH 2.5)孵育2分钟来洗脱抗体。
  10. 孵育时,向试管中加入75μl-100μl2 M Tris-HCl(pH 8.5)。 在步骤11之后,体积应允许最终浓度为150μMTris。
  11. 2分钟后,将1-2ml来自步骤9的1-2ml洗脱的抗体在甘氨酸中添加到来自步骤10的管中。Tris(pH8.5)应当中和酸性甘氨酸。
  12. 重复步骤9-11三次。
  13. 用PBS(pH7.4)透析级分并测定抗体浓度,作为您的常规蛋白质工作
  14. 测试与西方,免疫沉淀和免疫荧光的抗体。

食谱

  1. 10x TBS(1 L)
    24.23克Tris
    80.06克NaCl
    在800 ml水中混合
    用盐酸将pH调至7.6 将音量调节为1 L.
  2. TBST(1 L)
    100ml 10×TBS
    900毫升超纯水
    500μlTween 20
    注意:吐温20非常粘稠,会粘在您的测量移液管的尖端。 确保你拿到正确的数量,通过上下移动添加缓冲区。 同时,缓冲液应搅拌。
  3. PBS(pH7.4)(1L) 8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    800ml H 2 O 2 / 将pH调节至7.4 用另外蒸馏的H 2 O调节体积至1L 高压灭菌
    灭菌
  4. Ponceau S溶液(1 L)
    1 g Ponceau S
    50ml乙酸
    将H <2> O添加到1 L

致谢

该方案改编自Kurien(2009),并在美国加利福尼亚州斯坦福大学生物科学系Guowei Fang的实验室开发。 NIH的资助支持了这项工作。

参考文献

  1. Kurien,B.T。(2009)。 从硝化纤维素蛋白质印迹上切下的抗原条带中亲和纯化自身抗体 Methods Mol Biol 536:201-211。
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How to cite this protocol: Fang, L. (2012). Antibody Purification from Western Blotting. Bio-protocol 2(6): e133. DOI: 10.21769/BioProtoc.133; Full Text



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5/26/2017 1:17:43 AM  

Ming-Chieh Lin
National Taiwan University

The protocol is very easy to follow and my antibody works well after affinity purification, thanks for sharing. My question is, after the elution step, the antigen strips can reuse right? how many times can the strips be re-binded with serum and repeat the elution steps? I kept the serum flow through after first binding (9mL serum+2 strips) and I want to capture rest antibodies retain in the serum flow through. Thank you.

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7/12/2011 5:16:57 PM  

Yuanqing Lin
Test institute

I have problms with the elution of the antibodies. I checked with a second antibody if those from the serum are still on the menbrane and they are still there. All of them. I don't get a signal on WB with the eluate, and BCA neither shows higher concentration of protein in comparison to elution buffer with Tris. I variated the elution temperature (0- 25 °C) but I am still gtting the same result...
Do you have a solution for my problem?

8/31/2011 4:01:38 PM  

bio-protocol

"BCA neither shows higher concentration of protein in comparison to [elution buffer with Tris]. "

Did you elute with Glycine or Tris? Tris will not elute the protein at all. If you used Glycine, have you checked the pH which is critical for the elution.

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