September 2013 – Bio-Resource

Immunoprecipitation Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples. Immunoprecipitaion in general involves the following Steps: Incubate sample with antibody against protein of interest. Separate antibody-protein complex from remaining sample Analysis Basic Principle Of Immunoprecipitaion Co-Immunoprecipitation: A protein complex can be isolated from a protein mixture by using an antibody that is specific for one protein of the complex. Applications of Immunoprecipitation Technique Isolate / Detect Proteins of interest Enrichment of low abundant proteins Study protein-protein interaction and protein complexes Identify unknown proteins in a protein complex Verify protein expression in a specific tissue. Immunoprecipitaion Procedure / Protocol Immunoprecipation Procedure / protocols involves the following steps Sample Preparation Use of an Isolate control Pre-Cleaning of sample Antibody Incubation Precipitation of Protein / Protein Complex Washing Elution Analysis of the Precipitate Lets look into each Step: Sample Preparation Samples used for immunoprecipitaion can be of any samples of biological origin. Lysis Buffer: Choice of buffer depends on goal of the immunoprecipitation experiment Lysis Buffer Criteria Concentration Range Type and amount of detergent Non-Ionic : 0.1 – 2.0 % Ionic: 0.01% to 0.5% Amount of Salt 0-1 M Presence of EDTA 0-5MM pH 6.0 – 9.0 Lysis Buffer should always contain protease inhibitors and phosphatise inhibitors Store lysates at -20dc or -80dc Avoid freeze thaw cycles Isotype Control Isotype control is used to establish the specificity of the signal and the amount of non-specific background. It should be done simultaneously with the IP antibody but in a different tube. Pre-Clearing This step is done to avoid any non-specific binding and thereby avoiding the background signal. This step helps to reduce the background and improve signal to noise ratio. It is an optional step to improve the signal. Antibody Incubation Amount of antibody required need to be find out and it is important to have optimal antibody to protein ratio to have better results. Different antibody to protein concentration ratios can be tried out (1:100 to 1:1000), Antibody incubation is done by incubating the IP antibody with the lysate by gentle agitation, it can be done at room temperature for 2hrs or at 4dc overnight . Incubation time and antibody concentration need to be optimized for better results. Precipitation of Protein / Protein Complex Protein A,G or L coupled to beads (Aarose or Sepharose) are most commonly used for Protein precipitation. Base on the host species and the type IP antibody beads can be selected. Antibody can be directly conjugated to the beased the advantage of this is that of having lesser non-specific bands. Immunoprecipitation is done by incubating with the antibody and then centrifuging it at 4dc. Washing Washing is done to remove the non-specifically bound proteins from the immunoprecipitate. Washing is generally done with Lysis buffer or PBS. PBS is less stringent and can be used for analysis of protein-protein complexes. Elution Elution step is to dissociate the specifically bound proteins from antibody-bead complex. Elution Buffers: 2X Laemmli Buffer: Harsh Buffer can denature protein Glycine Gradient (upto 1M): More gentle can dissociate protein of interest without eluting IP antibody. Analysis of the Precipitate: Analysis can be done using the following methods SDS – PAGE Western Blotting Gel band Excision and sequencing Mass Spectrometry Scintillation counter or X-ray film for radioactive samples, etc. References: Abcam Technical Resources Thermo-Scientific Technical Resources Got something to say about this post? Leave a comment...your comments are valuable for improving the posts.

Month: September 2013

Immunoprecipitation: Procedure, Analysis and Applications

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