Immunoprecipitation Presentation

Definition, Principle, Types, Methods and Applications of IMMUNOPRECIPITATION (Immunology)

1. IMMUNOPRECIPITATION S UBMITTED BY : DAV COLLEGE, Vibhuti Sardana CHANDIGARH Manisha (SEC-10)

2. CONTENTS : i ) INTRODUCTION ii) PRINCIPLE iii) PROCEDURE iv) TYPES v ) METHODS vi) APPLICATIONS

3. INTRODUCTION Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. It provides a sensitive assay for the presence of a particular antigen, in a cell or tissue .

4. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins. It is widely used method for protein isolation from complex samples such as cell lysates , serum and tissue homogenates. By targeting a known protein with a specific antibody, it may be possible to pull the entire protein complex out of solution. It is used to purify and enrich the protein of interest from a complex mixture. IP is an important step in many proteomics studies .

5. PRINCIPLE The cardinal principle behind immunoprecipitation (IP) is the isolation of an antigen by interaction between the antigen and a specific antibody conjugated to a  sedimentable matrix . Antigen source may vary from cells or tissues without labeling, radio labeled or tagged cells, labeled or unlabeled proteins isolated from subcellular fractionation or in-vitro translated proteins.

6. Immunoprecipitation can also be helpful in protein fraction analysis separated by gel filtration or differential sedimentation based on density gradients. In order to capture interacting antigens , antibodies developed from various animal species that could be either polyclonal or monoclonal , are used for immunoprecipitation . These antibodies are tagged  non-covalently with specific immunoadsorbents such as  protein A–or   protein G agarose  or covalently linked with a solid-phase matrix.

7. PROCEDURE Immunoprecipation Procedure/Protocols involves the following steps: i ) Sample Preparation ii) Use of an Isolate control iii) Pre-Cleaning of sample iv) Antibody Incubation v) Precipitation of Protein / Protein Complex vi ) Washing vii ) Elution viii ) Analysis of the Precipitate

8. STEP 1 -: 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.

9. STEP 2 -: 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. STEP 3 -: 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.

10. STEP 4 -: 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.

11. STEP 5 -: Precipitation of Protein/Complex • Protein A,G or L coupled to beads ( Agarose 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 bead, 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. STEP 6 -: 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.

12. STEP 7 -: 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.

13. STEP 8 -: 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. 

14. TYPES OF IMMUNOPRECIPITATION

15. Individual Protein Immunoprecipitation(IP) :  It involves using an antibody that is specific for a known protein to isolate that particular protein out of a solution containing many different proteins.  These solutions will often be in the form of a crude lysate  of a plant or animal tissue.  Other sample types could be body fluids or other samples of biological origin.

16. Protein Complex Immunoprecipitation (Co-IP):  The immunoprecipitation of intact protein complexes is known as co- immunoprecipitation (Co-IP).  Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins , which is also called protein-protein interactions .  Main purpose of Co-IP is the identification of interaction partners (other proteins, ligands , co-factors, or signalling molecules) to the protein of interest.

17. Chromatin Immunoprecipitation ( ChIP ):  Chromatin immunoprecipitation ( ChIP ) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest.  This technique gives a picture of the protein-DNA interactions that occur inside the nucleus of living cells or tissues.

18. RNP Immunoprecipitation (RNPs):  Live cells are first lysed and then the target protein and associated RNA are immunoprecipitated using an antibody targeting the protein of interest. It targets RNA-binding proteins ( ribonucleoproteins ).  RNA-protein complexes are separated by RNA extraction.

19. METHODS OF IMMUNOPRECIPITATION There are basically 2 methods of immunoprecipitation : 1.) DIRECT METHOD ( Pre- I mmobilized A ntibody A pproach ):  Antibodies that are specific for a particular protein (or group of proteins) are immobilized on a solid-phase substrate such as  superparamagnetic microbeads  or on microscopic  agarose  (non-magnetic) beads.  The beads with bound antibodies are then added to the protein mixture, and the proteins that are targeted by the antibodies are captured onto the beads via the antibodies; in other words, they become immunoprecipitated .  This method is preferred when the target protein is abundant. Also, in this method, less primary Ab is required. Analyse precipitated antigen

20. 2.) INDIRECT METHOD ( Free Antibody Approach ):  Antibodies that are specific for a particular protein, or a group of proteins, are added directly to the mixture of protein.  The antibodies have not been attached to a solid-phase support yet.  The antibodies are free to float around the protein mixture and bind their targets. As time passes, beads coated in PROTEIN A/G are added to the mixture of antibody and protein.  At this point, the antibodies, which are now bound to their targets, will stick to the beads.  This method is preferred when the Ab has poor affinity for the target or when the target protein is in low abundance. Analyse precipitated antigen

21. APPLICATIONS • It is used to estimate the molecular weight , identity or quantity of a protein of interest. • Study protein- protein interaction . • Determine the concentration of protein . • Analyse the expression level of protein of interest . • Studying Cancer development , cell signalling pathway and diseases . • To determine post translational modifications . • Verify protein expression in a specific tissue. • Enrichment of low abundant proteins .

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