Immunoprecipitation
Immunoprecipitation is a precipitaion technique which allows the isolation
of protein or protein complex from biological samples.
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.
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
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
inhibitors
Store lysates at -20dc or
-80dc
-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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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