Immunoprecipitation (IP) is a powerful method in molecular biology and biochemistry that involves the identification and isolation of particular proteins from a solution, tissue or lysate by employing the use of antibodies specific to the protein of interest. The process of IP has been employed by researchers to develop a number of experimental and analytical techniques that involve the identification of proteins and their interactions with other proteins or with nucleic acids, reviewed by (Jackson and Dickson, 1999; Ponzielli et al., 2008; Yaciuk, 2007). Such IP techniques have allowed researches to investigate the interactions of biologically active proteins such as receptors and their ligands or kinases and phosphatases with their substrates and have also provided information about the interaction of transcription factors with DNA.
Further techniques have been employed within a pharmacological context in order to diagnose illness and identify drug targets. The presence or absence of a particular protein or family of proteins can often be an excellent indicator of disease pathology and prognosis and therefore the identification of such proteins by IP is an important procedure in diagnosing and treating many human diseases and illnesses, including certain types of cancer, reviewed by (Liu, 2004; Liu, 2005).
The methods and techniques of immunoprecipitation will be described here. Both historical and current methods of IP will be investigated, in the context of molecular biology and biochemical research and in terms of pharmacology will be discussed. Studies that have employed immunoprecipitation to identify important interactions from the point of view of human disease will be investigated.
History and current developments in immunoprecipitation
Early methods of immunological identification of proteins enabled the source of a protein such as ferritin (an intracellular iron storage globular protein complex) to be differentiated in terms of the species they were derived from, such as humans, horses and dogs, depending on their immunoprecipitation with serum from each species, (Mazur and Shorr, 1950), reviewed by (Richter, 1973). Since the 1950s, many methods of immunoprecipitation have been developed and are used widely in molecular biology and biochemistry research and in pharmacology. The history and development of such techniques and modern methods of immunoprecipitation will be discussed below.
Methods of Immunoprecipitation
According to (Kaboord and Perr, 2008), the methods of IP can be divided into three categories. Each with its own particular advantages: traditional (classical) method, oriented affinity method and direct affinity method. All three require a substance known as the immunomatrix to which antibody and/or the target protein is bound. In the traditional and oriented affinity methods, the immunomatrix that is typically used is agarose beads or resin that can be either protein A or protein G, reviewed by (Kaboord and Perr, 2008).
Protein A and Protein G are immunoglobulin antibody binding Staphylococcal and Streptococcal bacterial proteins respectively, reviewed by (Kaboord and Perr, 2008). Protein A and Protein G differ in their ability to bind antibodies so both tend to be used in IP to maximise the chances of success, reviewed by (Kaboord and Perr, 2008).
A number of IP kits are available commercially to researchers who are looking to carry out various types of immunoprecipiation and immunoblotting procedures. Such kits include the necessary immunomatrix and binding proteins and are available from such suppliers as Pierce and Thermo Scientific, Sigma-Aldrich and Clontech.
Although immunological techniques involving gel electrophoresis are not always considered to be immunoprecipitation per se, such techniques involve the extraction of protein from solution, lysate or tissue by subjecting such samples to an electric current rather than using an immunomatrix but do involve the identification of specific proteins using antibodies, so such gel-based techniques will be considered as immunoprecipitation for the purposes of this review. IP is often followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting or western blotting in order to identify specific proteins by antibody incubation from a number that may have been identified or “pulled down” by immunoprecipitation.
The advantages and disadvantages of two gel-based immunological techniques were compared in a review article published in the late 1980s (Bjerrum and Heegaard, 1989). Immunoelectrophoresis involves separating an antigen mixture by gel electrophoresis, followed by diffusion of antibodies into the gel in order to identify specific proteins. Immunoblotting or western blotting involves transferring protein separated by SDS-PAGE onto a nitrocellulose or polyvinylidene fluoride (PVDF) membrane followed by the use of a specific primary antibody against the protein of interest and a secondary fluorophore-linked antibody to highlight the presence and size of the protein under investigation, reviewed by (Bjerrum and Heegaard, 1989).
Although the review of Bjerrum and Heegaard (1989) determined that both immunoelectrophoresis and immunoblotting have their own advantages and that these two techniques should be used in a complimentary fashion, over the last twenty years western blotting has been preferred and utilised more frequently by researchers than immunoelectrophoresis due to immunoblotting allowing the molecular weight of proteins to be determined, reviewed by (Dickson, 2008).
Antiangiogenic drugs including (Z)-3-[4-(dimethylamino)benzylidenyl]indolin-
2-one (a growth factor receptor and kinase inhibitor) and oxindole (a kinase inhibitor) were successful in the treatment of vascularised tumours in rats (Jeffes et al., 2005). This study involved the use of the enzyme-linked immunoadsorbant assay (ELISA) to analyse growth factors. Glioma cells were identified by IP by using antibodies against the expression of antigenic growth factor receptors.
IP was used to identify the presence of Yo antibodies in ovarian and breast cancer patients (Monstad et al., 2006). Yo antibodies are onconeural antibodies specific for cdr2 antigens expressed in neurons and are specifically associated with the presence of tumours (Monstad et al., 2006). Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD), a condition that occurs in breast and ovarian cancer patients.
IP is dependent on the position of the epitope specific for the antibody being present at the exterior of the protein of interest. In this way, IP has a drawback in that not all proteins can be pulled-down, depending on their conformation. Proteins are often found bound in large complexes and IP can therefore result in the isolation of several proteins rather than the specific protein of interest. For this purpose, secondary techniques are required to specifically isolate the protein of interest.
Whereas the typical methods of immunoprecipitation involve the investigation of protein-protein interactions, chromatin immunoprecipitation (ChIP) involves the study of interaction of proteins with DNA, allowing the identification of proteins whose function is to interact with nucleic acids. There are four major steps in the protocol for ChIP and they are fixation, sonication and the IP itself that is followed by analysis of data, reviewed by (Das et al., 2004) (Dasgupta and Chellappan, 2007).
C hIP involves treatment with formaldehyde in order to generate DNA-protein cross links and can be used to map the genomic locations of post-translationally modified histones, reviewed by (Clark and Shen, 2006) transcription factors, reviewed by (Stewart et al., 2006) and chromatin modifying enzymes, reviewed by (Collas and Dahl, 2008). ChiP can identify protein-protein and DNA-protein interactions and also identifies where in the genome this happens, reviewed by (Aparicio et al., 2005; Aparicio et al., 2004).
ChIP technology has been employed to investigate the proteins interactions of the small GTPase Ras, reviewed by (Dunn et al., 2005) and the role of this important protein in the mitogen-activated protein kinase pathway in breast cancer (Espino et al., 2006), and the role of Ras and its associated protein complex in its interactions with specific transcription factors, also in cancer (Sreeramaneni et al., 2005).
RNA precipitation (RIP) is similar to ChIP, except that in the case of RIP, the interactions of protein with RNA are investigated rather than protein interactions with DNA and involves generating cross-links between the RNA and the protein by formaldehyde treatment, followed by the identification of cross-linked RNA and protein by IP, reviewed by (Gilbert and Svejstrup, 2006).
A similar and related technique known as nucleosome IP that is used for the identification of nucleosomes has been recently developed in order to identify specific nucleosomes that are responsible for the specific post-translational modifications of proteins, reviewed by (Clark and Shen, 2006).
Co-immunoprecipitation (Co-IP) is used to identify and isolate proteins in complex, reviewed by (Masters, 2004), (Free et al., 2009), (Yaciuk, 2007), (Anderson, 1998). Co-IP has been used in studies of cancer biochemistry by investigating the interaction of tumour suppressor proteins (Su, 2003). The role of the small GTPase R-Ras in cancer has been investigated by Co-IP, regarding its role in cancer and specifically its interaction with transcription factors (Xu and Komatsu, 2009).
A comparison of the use of immunoprecipitation techniques in pharmocology
Immunoprecipitation has been used by researchers in molecular cell biology and pharmacology to identify the interactions of many different proteins. To compare and contrast the various IP techniques used by researchers, three different studies that all had a similar aim in identifying small GTPases and other signalling proteins from hepatic tissues will be discussed.
An early study, following the identification of the small GTPase Ras as an oncogene in hepatomas seeked to identify the location of mutations in the Ras oncogene that were responsible for tumour formation (Wiseman et al., 1986). Immunoprecipitation was employed in order to identify Ras proteins in a cell line treated with carcinogens. A monoclonal antibody specific to Ras was used in the immunoprecipitation study to isolate and pull down Ras from the treated cells, according to the protocol of a previous study that sought to identify oncogenes involved in liver cancer (Reynolds et al., 1986). In this method, NIH.3T3 cells were metabolically radiolabelled with S35 Methionine, extracted and cleared by centrifugation. The cell lysates were then incubated with the specific monoclonal antibody and the immunoprecipitation was performed by coating a protein-sepharose matrix with anti-rat immunoglobulin-G in order to detect the primary monoclonal antibody by the formation of immunocomplexes. To identify the bound proteins, the samples were then subjected to SDS-PAGE electrophoresis, the gel was dried and subsequently exposed to X-ray film in order to visualise the S35 label of the immunoprecipitated proteins of interest.
A recent study has employed an alternative method of immunoprecipitation in order to identify hepatic proteins present in liver cancer by tagging Lithocholic acid (LCA), a type of bile acid (Ikegawa et al., 2008) . Antibodies were generated using the the 3a-hydroxy-5ßsteroid moiety of LCA in rabbits using an immunogen of LCA bound to BSA. Hepatic proteins were tagged with LCA and were recognized by the generated antibodies, which therefore allowing their identification by immunoprecipitation (Ikegawa et al., 2008). The proteins identified in this study by included the small GTPase signalling proteins Rab and Ras (Ikegawa et al., 2008).
The presence of Ras and Rab proteins in the liver is pharmacologically relevant because such small GTPase proteins, have been identified as oncogenes (Reynolds et al., 1986) and when bound to LCA, have been implicated in liver toxicity and DNA damage, that can lead to cancer (Gelb et al., 1982), (Fickert et al., 2006).
In order to begin the study, antibodies were generated using LCA bound to BSA using a 6-aminohexanoic acid and succinic acid spacer (LCA-6AH) as the immunogen or hapten. The LCA-6AH was then subjected to a number of steps in order to produce the final hapten LCA-HS, which in turn was coupled to BSA to produce LCA-BSA conjugates.
These LCA-BSA conjugates were then sub-cutaneously injected with Freund’s complete adjuvant into rabbits and the levels of antibody in the rabbit subjects was analysed throughout the study by ELISA. Competitive ELISA was used to determine the binding ability of all anti-LCA antibodies that were produced. It was shown that antibodies had the highest affinity for LCA when compared to other bile acids.
The ability of the antibody was then assayed by ELISA to investigate its ability to bind LCA residues on proteins by studying the ability of the antibody to recognize LCA residues bound to ovalbumin. Ikegawa et al (2008) showed that the antibodies could bind to protein-bound LCA and therefore that it was likely that the antibodies could be successfully used in immunoblotting.
MALDI-TOF-MS (Matrix-assisted laser desorption/ionization-time of flight-mass spectrophotometry) analysis was performed to identify individual peptides to which the antibody was bound and therefore provided insights into how specific the produced antibody actually was by investigating binding to lysozymes. The MALDI-TOF-MS analysis performed by Ikegawa et al (2008) identified several lysine residues on the lysozyme as the binding sites for LCA. Subsequent SDS-PAGE and immuoblotting confirmed that the antibody could specifically recognise LCA bound to the lysine residues of the lysozyme.
Having established that the identified antibody could be successfully used to in turn identify proteins bound to LCA, MALDI-TOF-MS analysis was again performed to establish the identity of the proteins bound to the LCA bile acid in liver samples taken from a rat that had undergone bile-duct ligation in order to produce an animal model to simulate cholestatic liver disease.
Subcellular compartments in the liver samples were identified by centrifuge fractionation and using this method, it was determined that the proteins which bound to LCA were soluble and localised to the cytosol. Rab and Ras proteins were identified as binding to LCA in liver tissue by immunoprecipitation, and by 2D gel analysis and silver staining. The 2D gel separated proteins not only by their molecular weight in electrophoresis (the first dimension), but also by their pH (the second dimension). Three spots of protein expression were observed with specific immunoprecipitate by 2D gel analysis and these spots where then analysed by MALDI-TOF-MS in order to specifically identify their protein content.
A further study investigated the effects of ethanol exposure on the distribution of GTPases in rat hepatocytes and employed immunological techniques in order to do so (Larkin et al., 1996). This study also used radiolabelling to tag specific proteins and centrifugation in order to separate cellular fractions. This study employed a protein sepharose-A beads rather than a gel matrix and anti-IgA immunoglobulins to trap specific proteins. Detection was again performed by SDS-PAGE and exposure to X-ray film, similar to that described by (Reynolds et al., 1986).
The comparison of these three immunoprecipitation protocols to investigate similar problems emphasises how techniques in IP have developed over the last twenty years, but also highlights that the IP method is as relevant today for studying pharmacologically important proteins and their functions in cancer. (Reynolds et al., 1986) utilised a simple technique in order to identify and pull down a specific protein from cell lysates, while (Larkin et al., 1996) employed similar techniques to identify proteins affected by exposure to ethanol and (Ikegawa et al., 2008) have used a refined protocol in order to more specifically identify the proteins of interest, but the principles of each study remain consistent.
MALDI-TOF-MS now allows the identification of many individual proteins by their mass by (Ikegawa et al., 2008), while SDS-PAGE, a less specific technique requiring an antibody to each individual protein was the only technique of identification available to (Reynolds et al., 1986).
Immunoprecipitation techniques are used to investigate the presence of proteins, the interactions of proteins with other proteins and also their interactions with DNA. There are a number of methods of performing the procedure depending on the aims of the study and the type of proteins under investigation.
Immunoprecipitation has been used to investigate the role of small GTPase proteins in liver disease and because the proteins investigated by IP methods are often involved in human diseases, immunoprecipitation is a very important and powerful technique in pharmacology for the identification of drug targets.