为了使您在Abcam官网的浏览体验更顺畅,请使用最新版本的浏览器比如 Google Chrome

致电 +86 21 2070 0500 或 联系我们

  • 我的账户
  • 退出
登录 或 注册

欢迎光临

登录 或

您还没有帐户?

注册
我的购物篮
快速订购
Abcam homepage

  • 研究用途的产品
    按产品类型
    一抗
    二抗
    免疫测定试剂盒与试剂
    细胞与组织成像工具
    细胞与生化测定
    蛋白质与肽
    按产品类型
    蛋白质组学工具
    激动剂、活化剂、拮抗剂与抑制剂
    细胞株和裂解液
    多重miRNA检测
    多因子分析试剂盒
    按研究领域
    癌症
    心血管
    细胞生物学
    表观遗传学
    代谢
    发育生物学
    按研究领域
    免疫学
    微生物学
    神经科学
    信号转导
    干细胞
  • 定制化产品 & 合作关系
    定制化产品 & 合作关系

    定制化产品和商务合作加速您的诊断和治疗项目

    定制化产品

    与我们成为伙伴

  • 技术支持
    支持中心

    获取研究建议和支持

    查看支持中心

    Protocols

    一步一步完成您的实验

    查看Protocols

  • 活动通知
    • 会议年历
    • 肿瘤研究
    • 心血管研究
    • 染色质及细胞核信号转导研究
    • 免疫学
    • 神经生物学
    • 干细胞研究
    • 商品展览
    • 实验技术视频
    查看最新活动

    活动说明、摘要提交、演讲者、注册方式及更多信息

    查看全球活动安排

  • 通路
    细胞信号转导通路

    查看所有通路

    查看交叉通路

产品: 类別
  • 肿瘤研究
  • 心血管研究
  • 细胞生物学
  • 染色质及细胞核信号转导研究
  • 发育生物学
  • 免疫学
  • 代谢
  • 微生物学
  • 神经生物学
  • 信号转导研究
  • 干细胞研究

Using IgM antibodies for IP

Tips on immunoprecipitation procedures using IgM antibodies

View a pdf version of this page PDF

IgM antibodies are difficult to use in immunoprecipitation due to its pentameric structure. IgM antibodies also do not bind well to protein A or protein G. There are two alternative methods available:

1. Protein L beads

Protein L is a 36 kDa immunoglobulin binding protein which can be purified from peptostreptococcus magnus. Protein L binds antibodies through light chain interactions (whereas protein A and G bind the Fc heavy chain region of antibodies). Protein L binds a wider range of antibody classes than protein A or G because no part of the heavy chain is involved in the binding interaction. It is able to bind all antibody classes, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to protein L.

Binding of protein L to immunoglobulins is restricted to those that contain kappa (K) light chains (i.e. K chain of the VL domain). In humans and mice, kappa (K) light chains predominate. The remaining immunoglobulins have lambda (λ) light chains which will not bind protein L. Furthermore, protein L is effective in binding only certain subtypes of kappa light chains. For example, it binds human VKI, VKIII and VKIV subtypes but does not bind the VKII subtype. Binding of mouse immunoglobulins is restricted to those having VKI light chains.

There are several advantages to using protein L:

  1. Protein L does not bind to bovine, sheep or goat antibodies, making it ideal for purification of mouse IgM from cell culture media containing bovine serum.
  2. Protein L binds to kappa light chains on antibodies from a wide range of species without interfering with antigenbinding sites, making it very suitable for immunoprecipitation using IgM antibodies.
  3. It binds to all classes of Ig (e.g. IgG, IgM, IgA, IgE and IgD) making it particularly useful for IgM purification which cannot be obtained using protein A or G.

2. Use an IgG anti-IgM antibody on protein A or protein G beads.

This method involves coupling an IgG anti-IgM antibody to protein A or G beads (see method below). These beads can then be used in the normal immunopreciptiation procedure using the IgM antibody. The IgM will bind to the beads by binding to the anti-IgM antibody.

The procedure below describes how to couple the anti-IgM antibody to the protein A or G coated beads using dimethylpimelimidate (DMP):

Reagents:

  1. Borate buffer 200 mM + NaCl 3 M pH 9.0
  2. Dimethylpimelimidate (DMP) 20 mM in 200 mM borate buffer + 3 M NaCl, pH 9.0
  3. Ethanolamine 200 mM pH 8 (1:80 dilution of stock ethanolamine)
  4. Phosphate buffered saline (PBS)
  5. PBS + sodium azide 0.1%
  6. Glycine 200 mM pH 2.5
  7. Protein A or G beads (sepharose)

Method:

We recommend using 2 mg of antibody per ml wet beads (use appropriate antibody/protein A or G combination).

  1. Add 250 mg Protein A or G sepharose beads (beads) to 2 ml PBS + 0.1% sodium azide, for one hour before use. This allows the beads to swell.
  2. Mix the beads with an appropriate concentration of antibody for 2 hours at room temperature (using a rotator or roller).
  3. Centrifuge at 2500 rpm for 5 minutes.
  4. Wash the beads twice in 10X volume of 200 mM borate buffer + 3 M NaCl.
  5. Resuspend the beads in 20 mM DMP.
  6. Rotate / agitate the beads for 30 minutes at room temperature.
  7. Centrifuge the beads at 2500 rpm for 5 minutes.
  8. Wash the beads 2X in 200 mM borate buffer + 3M NaCl.
  9. Wash the beads 1X in 20 mM ethanolamine. This stops the coupling reaction.
  10. Resuspend the beads in 20 mM ethanolamine and rotate for 2 hours at room temperature.
  11. Centrifuge the beads at 2500 rpm for 5 minutes.
  12. Wash the beads 2X in PBS.
  13. Wash the beads 2X in 20 0mM glycine.
  14. Wash the beads 2X in PBS.
  15. The beads can be stored at 4oC in PBS + 0.1% sodium azide (1 X PBS to 1 X beads)
  16. These beads are now ready for use in immunoprecipitation with IgM antibody. From this point, proceed with the immunoprecipitation as described on our immunoprecipitation protocol page.

通过邮件收到学术资源和促销信息 注册
研究类别(A-Z)
  • 肿瘤研究
  • 心血管研究
  • 细胞生物学
  • 发育生物学
  • 染色质及细胞核信号转导研究
  • 免疫学
  • 代谢
  • 微生物学
  • 神经生物学
  • 信号转导研究
  • 干细胞研究
产品种类(A-Z)
  • 一抗
  • 二抗
  • 生化试剂
  • 亚类对照
  • 流式细胞仪多色选择器
  • 试剂盒
  • 内参对照
  • 细胞裂解液
  • 多肽
  • 蛋白酶
  • 组织切片
  • 蛋白标签及细胞标记物
  • 相关试剂及工具
帮助与支持
  • 技术支持
  • 技术FAQ
  • 实验步骤与建议
  • 购买FAQ
  • RabMAb 产品
  • 培训课程
  • 按靶标浏览
企业
  • 企业资讯
  • 投资者关系
  • 公司新闻
  • 招贤纳士
  • 关于我们
活动通知
  • 商品展览
  • 会议
全球网站
  • abcam.com
  • abcam.co.jp
QR code for Weechat

微信扫码 关注我们

沪ICP备14046373号-1

沪公网安备 31011502007413号

  • 销售条款
  • 网站使用条款
  • Cookie政策
  • 隐私权政策
  • 法律
© 1998-2023 艾博抗(上海)贸易有限公司版权所有