Immunoprecipitation is a precipitaion technique which allows the isolation of protein or protein complex from biological samples.
Incubate sample with antibody against protein of interest.
Separate antibody-protein complex from remaining sample
Analysis
2. 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
3. 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
4. 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 should always contain protease inhibitors and phosphatise inhibitors
• Store lysates at -20dc or -80dc
• Avoid freeze thaw cycles
•
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
•
10. 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.
11. 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.
