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Wednesday, September 25, 2013

Immunoprecipitation: Procedure, Analysis and Applications


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

Immuno precipitation


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

  1. Isolate / Detect Proteins of interest
  2. Enrichment of low abundant proteins
  3. Study protein-protein interaction and protein complexes
  4. Identify unknown proteins in a protein complex
  5. Verify protein expression in a specific tissue.
Immunoprecipitaion Procedure / Protocol

Immunoprecipation Procedure / protocols involves the following steps
  1. Sample Preparation
  2. Use of an Isolate control
  3. Pre-Cleaning of sample
  4. Antibody Incubation
  5. Precipitation of Protein / Protein Complex
  6. Washing
  7. Elution
  8. 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
  1. SDS – PAGE
  2. Western Blotting
  3. Gel band Excision and sequencing
  4. Mass Spectrometry
  5. Scintillation counter or X-ray film for radioactive samples, etc.



References:

Abcam Technical Resources
Thermo-Scientific Technical Resources