蛋白质组学

免疫共沉淀和免疫沉淀的图示和实验步骤

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免疫沉淀(IP)是一种利用的抗原 抗体反应的原则,确定具体反应中混合物的抗体蛋白数量及具体物理特性的方法。而免疫共沉淀(Co-IP) 则作为分析蛋白质间的相互作用而被广泛认知。下面是这两种实验方法的具体操作步骤。

Steps of IP:

1. Form the antigen-antibody complex (immune complex) by incubating specific antibody with the antigen-containing sample for 1 hour to several hours.

2. Capture the immune complex on an immobilized Protein A or Protein G agarose gel support by incubation for 0.5-2 hours.

3. Remove any non-bound protein (non-immune complex sample components) from the precipitated complex by washing gel support with additional sample buffer.

4. Boil gel support in reducing SDS-Page sample loading buffer.

5. Recover sample eluted in loading buffer from gel support and analyze by sds-page .

6. Perform Western blot analysis, probing with antigen-specific antibody.

Steps of coIP:

1. Lyse cells and prepare sample for immunoprecipitation.

2. Pre-clear the sample by passing the sample over beads that are not coated with antibody to soak up any proteins that non-specifically bind to the beads.

3. Incubate solution with antibody against the protein of interest. Antibody can be attached to solid support before this step (direct method) or after this step (indirect method). Continue the incubation to allow antibody-antigen complexes to form.

4. Precipitate the complex of interest, removing it from bulk solution.

5. Wash precipitated complex several times. Spin each time between washes or place tube on magnet when using superparamagnetic beads and then remove supernatant. 6.After final wash, remove as much supernatant as possible.

7. Elute proteins from solid support (using low-pH or SDS sample loading buffer).

8. Analyze complexes or antigens of interest. This can be done in a variety of ways:

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by gel staining.

SDS-PAGE followed by: staining the gel, cutting out individual stained protein bands, and sequencing the proteins in the bands by MALDI-Mass Spectrometry

Transfer and Western Blot using another antibody for proteins that were interacting with the antigen followed by chemiluminesent visualization.

免疫共沉淀的优点是可以得到自然状态下的蛋白质互作产物,有效避开人工因素的影响。不过由于该实验需事先对所测蛋白进行预测,如果预测不成功,很有可能整个实验就得重做了。

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