Immunoprecipitation Protocol (PDF)
Introduction
Immunoprecipitation involves the interaction between a protein and its specific antibody, the separation of these immune complexes with Protein G or Protein A, and the subsequent analysis by SDS-PAGE. This technique provides a rapid and simple means to separate a specific protein from whole cell lysates or culture supernatants. Additionally, one can use immunoprecipitation to confirm the identity or study biochemical characteristics, post-translational modifications, and expression levels of a protein of interest. The procedure can be divided into several stages:
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Sample preparation
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Preclearing
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Antibody incubation/formation of antibody-antigen complexes
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Precipitation
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Analysis by SDS-PAGE
The protocol below offers a general guideline for immunoprecipitation. Optimization may be required for each specific antigen and antibody. The abundance of a given protein in a sample is variable and a critical factor for obtaining desired results. In this protocol, a cell concentration of approximately 107 cells/ml is recommended as a starting point. Fine adjustments to cell concentrations and volumes of cell lysate used in each immunoprecipitation experiment may be necessary.
The choice of lysis buffer is critical and dependent on the nature of the protein to be studied. NP-40, a non-ionic detergent, is the most commonly used detergent in cell lysis buffers. Increasing the salt concentration, decreasing the detergent concentration, or changing the detergent to Triton X-100, Saponin, Digitonin, CHAPS etc. are steps that can be taken to optimize conditions for immunoprecipitation.
The preclearing step is incorporated into the procedure to lower the amount of non-specific contaminants in the cell lysate and to remove proteins with high affinity for Protein G or Protein A. Many investigators choose to skip this step in cases where the protein of interest exists abundantly in the sample.
The success of immunoprecipitation depends on the affinity of the antibody for its antigen as well as for Protein G or Protein A. In general, while polyclonal antibodies are best, purified monoclonal antibodies (mAb), ascites fluid, or hybridoma supernatant can also be used. Some mAbs work very well, whereas others may give unsatisfactory results. The eBioscience antibodies that have been reported in the literature to work for immunoprecipitation are listed for this application. It is recommended that the specified experimental conditions be followed as published; however, optimization of condition by the investigator may be necessary.
The strength of interaction between the mAb and Protein G or Protein A is an important factor in the decision of which slurry to use. Protein G coupled to some insoluble matrix (e.g. sepharose beads) binds well to most subclasses of rat immunoglobulins and mouse IgG1, while Protein A binds much better to mouse IgG2a, IgG2b, and IgG3.
Table 1: Binding Characteristics of Some Immunoglobulins
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Binding Characteristics of Some Immunoglobulins
|
| Immunoglobulin |
Protein A |
Protein G |
| Mouse IgG1
|
+
|
++
|
| Mouse IgG2a
|
+++
|
+++
|
| Mouse IgG2b
|
++
|
++
|
| Mouse IgG3
|
+
|
+++
|
| Mouse IgM
|
-
|
-
|
| Mouse IgA
|
-
|
-
|
| Mouse IgE
|
-
|
-
|
| Rat IgG1
|
+
|
+
|
| Rat IgG2a
|
-
|
+++
|
| Rat IgG2b
|
-
|
++
|
| Rat IgG2c
|
+
|
++
|
| Human IgG1
|
+++
|
+++
|
| Human IgG2
|
+++
|
+++
|
| Human IgG3
|
-
|
+++
|
| Human IgG4
|
+++
|
+++
|