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

我们使用cookies,使我们网站尽可能对您有帮助。

我们的Cookie政策介绍了您如何能够取消cookie使用。

如果您不改变cookie设置,我们认为您愿意接受cookie。

继续 继续

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
  • 诊断和治疗的解决方案
    定制方案及合作关系

    定制抗体开发及商务合作助力您的诊断和治疗探索。

    与我们一起开创定制方案

    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

在新冠疫情期间支持我们的客户和员工–了解更多

Immunoprecipitation troubleshooting tips

Related

  • All IP resources
    • An introduction to immunoprecipitation webinar
      • Advanced immunoprecipitation webinar
        • Immunoprecipitation protocol
          • Cross-linking X-ChIP protocol
            • RNA immunoprecipitation (RIP) protocol
              • Primary antibodies for IP

                ​​Detailed troubleshooting for immunoprecipitation. 

                Print this protocol.

                High background

                Carry over of proteins that are not detergent soluble
                Remove supernatant immediately after centrifugations. This should leave insoluble proteins in the pellet. If resuspension occurs, centrifuge again.

                Incomplete washing
                Wash well at relevant stages by placing a lid on the tube and inverting several times before centrifuging.

                Non-specific proteins are binding to the beads
                Beads are not pre-blocked enough with BSA. Make sure BSA (fraction V) is fresh and incubate fresh beads for 1 hr with 1% BSA in PBS. Wash 3-4 times in PBS before using them.

                Antibody used contains antibodies that are not specific enough
                Use an affinity purified antibody, preferably pre-adsorbed.

                Too much antibody used leading to non-specific binding
                Check the recommended amount of antibody suggested. Try using less antibody.

                Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate
                ​Reduce the number of cells/lysate used. We recommend using 10-500 µg cell lysate.

                Non-specific binding of proteins to antibody
                If there are many proteins binding non-specifically, then try reducing the amount of sample loaded onto the beads. You can also pre-clear the lysate by pre-incubating the prepared lysate with the beads before commencing with the immunoprecipitation (please see the protocol). This should clear the lysate of any proteins that are binding non-specifically to the beads. Some researchers also use an irrelevant antibody of the same species of origin and same Ig subclass to pre-clear the lysate.

                Antigen degrading during immunoprecipitation
                ​Ensure fresh protease inhibitors are added when sample is lysed.

                High amount of antibody eluting

                Too much antibody eluting with the target protein
                Try reducing the amount of antibody. Crosslinking the antibody to the beads before the immunoprecipitation and eluting using a gentle glycine buffer gradient should significantly reduce the amount of antibody eluted.
                ​

                No eluted target protein detected

                Target protein not expressed in sample used/low level of target protein expression in sample used
                Check the expression profile of the target protein to ensure it will be expressed in the cells of your samples. If there is low level of target protein expression, increase the amount of lysate used. However, this may result in increased non-specific binding so it would be advisable to pre-clear the lysate before commencing with the IP procedure.

                Insufficient antibody for capture of the target protein
                Check that the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results.

                Target protein has not eluted from the beads
                Ensure you are using the correct elution buffer and that it is at the correct strength and pH for elution of the protein.

                Antibody has not bound to immunoadsorbant beads
                Ensure you are using the correct beads for the antibody isotype used.

                Incorrect lysis buffer used
                Check datasheet to see if the antibody detects denatured or native protein and ensure the correct lysis buffer is used. See our immunoprecipitation protocol.

                Protein of interest is obstructed by antibody heavy or light-chains

                Secondary antibody recognizes denatured/reduced primary antibody released during the IP procedure or endogenous IgGs
                Using secondary antibodies, recognizing the heavy and light-chain of the primary antibody for WB detection of IP samples will always result in two bands (the heavy-chain at 50kDa and the light-chain at 25kDa).
                To avoid interference by the antibody chains we recommend using secondary antibodies, such as  VeriBlot for IP, that specifically recognize native (non-reduced) primary antibodies.

                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
                © 1998-2021 艾博抗(上海)贸易有限公司版权所有