Protein analysis and puriﬁcation is aimed at elucidating the structure and function of proteins. In order to analyze a protein, you must have a reliable detection system that unambiguously enables you to follow the target protein. This is especially true when the target molecule is in crude or even semipuriﬁed form. The puriﬁcation of a bioactive molecule is frequently accomplished by using a deﬁnitive assay designed to recognize a property of the target protein. The protocols to be followed depend upon the starting material and on the analytical tools that are available. If you have a speciﬁc antibody, be it monoclonal or polyclonal, the procedures and analytical strategies you choose will differ from those that would be followed if the project is based on either a cloned gene or a bioassay. The ultimate goal is the same regardless of what you start with: to determine the structure and function of your molecule. In most cases, the availability of an antibody that recognizes the target protein will lead to a successful outcome. Many of the techniques that are described rely on a speciﬁc antibody to identify the target protein. Using the antibody, analytical reactions can be performed on a complex mixture and the effect on the target protein can be speciﬁcally followed. The antibody can also be utilized to screen an expression library, identify the bacterial colony expressing the target protein, and ultimately clone the gene. In the early stages of analysis when a biological activity is being followed, the active molecular agent may be a nucleic acid, protein, carbohydrate, lipid, any combination, or any other molecule such as a prostaglandin, leukotriene, or polyamine. In order to characterize the molecular nature of the active factor, one useful approach is to attempt to destroy the activity by treating it with an enzyme or subjecting the factor to a simple extraction procedure. For proteins, the extract is routinely treated with broad speciﬁcity proteolytic enzymes such as proteinase K, pepsin, pronase, and trypsin. It is important to include a suitable control to be sure that the treatment itself does not have an effect in the assay system being used. If the control is valid and the activity is destroyed, this constitutes strong evidence that the active fraction is proteinaceous. However, one must proceed cautiously. A negative result should not be viewed as absolute proof that the active factor is not a polypeptide. Proteins are often denatured by heating, resulting in the loss of bioactivity. If a solution of the active material is exposed to 56°, 65°C and, in the extreme, to boiling for times ranging from 3min to 1h and the bioactivity is destroyed, this is an indication that the active molecule is proteinaceous. Again, negative results should be interpreted with caution. Denaturing treatments such as heat or incubation with the anionic detergent sodium dodecyl sulfate (SDS) may cause the protein to partially lose its tertiary structure and open up, making it more susceptible to protease degradation. Inactivation of a biomolecule in a crude preparation by 65°C heat treatment may reﬂect an increased susceptibility to proteases more than heat lability. This should be kept in mind when performing early characterization experiments on the target molecule. There are many molecules that retain their bioactivity after boiling and protease treatment yet are made up partially or totally of protein. Some examples are glycoproteins, proteoglycans, and glycosaminoglycans.
Labeling Cells and Proteins
Sensitive and convenient analytical reactions have been devised to characterize proteins present as minor components of a complex mixture without going through a long, multistep puriﬁcation scheme. The availability of a speciﬁc antibody, either polyclonal or monoclonal, enables the enrichment and isolation of the target protein by immunoprecipitation. Biochemical reactions designed to analyze the target protein can be performed on the protein as part of the immunoprecipitated complex. The protein is then separated electrophoretically and analyzed. Alternatively, the target protein could be transferred and immobilized on a matrix such as nitrocellulose where additional analytical procedures may be performed. This chapter describes methods to tag the target protein, that is usually present as a minor component of a cell lysate, and then speciﬁcally follow it. General methods are presented for metabolically labeling live cells, and for labeling proteins located on the cell surface. In addition, the Chloramine T method of labeling a pure protein in solution is presented. There are two main approaches to radiolabeling cellular proteins. Each has certain advantages and limitations. In one approach, proteins present on the cell surface are labeled post-synthetically. Highest speciﬁc activities are achieved with radioiodination techniques. The other approach is the biosynthetic incorporation of radio-amino acids into the nascent polypeptide chain. Since the radiolabel is distributed throughout the polypeptide and all biosynthetic forms are labeled, this approach facilitates subsequent analysis of protein structure, such as peptide mapping, amino acid sequencing, and 2-dimensional electrophoresis.
Lysis: Preparation of the Cell-Free Extract
Following the labeling step, the cells are lysed and an extract is prepared. The conditions used for lysis should be gentle enough to retain the antibody binding sites but strong enough to quantitatively solubilize the antigen of interest. Important variables to consider are salt concentration, type and concentration of detergent, the presence of divalent cations, and pH. If a sample is to accurately reﬂect the state of the cells at the time of lysis, it will be necessary to choose a procedure that will eliminate the actions of proteolytic enzymes. Once the cells are lysed, proteolytic enzymes that were present in a compartmentalized state are released and can come in contact with the proteins in the extract. It is important to inactivate these proteases. The lysis buffer should contain salt concentrations between 0–1M, nonionic detergent concentrations between 0.1–2%, divalent cation concentrations between 0–10mM, EDTA concentrations between 0–5mM, and pHs between 6–9. In addition, an antiprotease cocktail should always be included. Some frequently used buffers are listed below.
- NP-40 lysis buffer: 50mM Tris-HCl pH 7.4–8.0, 150mM NaCl, 1.0% NP40 (or Triton X-100)
- High salt lysis buffer: 50mM Tris-HCl pH 7.4–8.0, 500mM NaCl, 1.0% Triton X-100
- Low salt lysis buffer: 50mM Tris-HCl pH 7.4–8.0, 10mM NaCl, 1.0% Triton X-100
- RIPAbuffer: 50mM Tris-HCl pH 7.4–8.0, 150mM NaCl, 1.0% Triton X-100, 0.5% Sodium deoxycholate (DOC), 0.1% SDS, 2mM EDTA
- General: 20mM Tris pH 8.0, 10mM NaCl, 0.5% Triton X-100, 5mM EDTA, 3mM MgCl2
Add an antiprotease cocktail made up of the reagents listed to the lysis buffer immediately before adding it to the cells. For phosphorylation studies, add the phosphatase inhibitors to attain the following ﬁnal concentrations: NaF 10mM and Na3VO4 1mM.
Principles of Immunoprecipitation
Antibodies as Detection Tools
Antibodies are exquisitely specific reagents that are used for identifying target proteins. Antibodies (Ab) consist of two types of polypeptide chains held together by disulfide bonds. The heavy chain has a molecular weight of roughly 55 kDa and the light chain, a molecular weight of approximately 25 kDa. Both heavy and light chains have a constant region and a variable region, both of which are located at the N terminus and which consist of about 100 amino acids. It is these regions that make contact with the antigen. The C-terminal constant region of the molecule is made up of a limited number of sequences and serves to define the antibody subtype. The five human isotypes of antibodies, IgM, IgD, IgG. IgA and IgE are defined by the five different heavy-chain types, mu (/L), Delta (&), gamma (‘y), alpha (a), and epsilon (e) respectively. There are two types of light chain, kappa (K) and lambda (A.) for all the immunoglobulin classes. IgG, the major immunoglobulin in serum, IgD and IgE are all composed of two heavy chains and two light chains. Structurally, IgM is a pentamer of IgG-like molecules while IgA is a dimer.
There are two types of antibodies used as reagents: monoclonal (MAb) and polyclonal. A polyclonal antibody solution is a heterogeneous mixture of antibodies that recognize numerous epitopes on a single antigen. The production of polyclonal antibodies, with the goal of producing a useful laboratory reagent, requires immunizing an animal with an extremely pure antigen. Specific antibodies are produced by many different clones of B-Lymphocytes, which react with different determinants on the same antigen molecule. This is the normal heterogeneous immunological response to an antigen which is referred to as polyclonal antibody. Therefore polyclonal antibodies are not suited for single epitope detection.
Kohler and Milstein (1975) developed methods that allowed for the growth of clonal cell populations that secrete antibodies of defined specificities. These cells are called hybridomas. The antibodies from a single isolated clone are identical (monoclonal) and will react with a specific epitope on the antigen against which it was raised. They can be propagated in vitro, will multiply indefinitely in culture, and will continuously secrete monoclonal antibodies with a defined specificity. A panel of monoclonal antibodies can be produced by a crude population of molecules and then undergo selection/screening on the basis of antigenic properties of the target molecule. Despite many similarities, each monoclonal antibody is a unique protein and presents its own purification challenges. MAbs differ in stability and affinity. These characteristics affect the behavior of individual MAbs in purification and analytical applications. The diversity of amino acids found in the variable region results in a wide range of isoelectric points for these molecules.
Antibody-Based Analytical Techniques: Western Blotting and Immunoprecipitation
Antibodies are commonly used to detect antigens in complex mixtures. Some well-known immunodetection methods include ELISA (enzyme-linked immunosorbent assay), double immunodiffusion, immunoprecipitation, and immunoblotting. If you are in possession of a specific antibody that recognizes your target protein, many analytical methods are possible. Western blotting and immunoprecipitation are two fundamental techniques that are used in association with polyacrylamide gel electrophoresis (PAGE) to identify and enrich proteins in a complex mixture so that they may be further analyzed. Western blots, dot blots, and colony/plaque lift all require immobilization of the target protein on the membrane. With Western blots, proteins were electrotransferred to a membrane after SDS-PAGE. For dot blots, nondenatured proteins are spotted directly onto a membrane. In colony/plaque lifts, intact bacterial colonies or phage plaques are transferred to a membrane and then lysed to expose the target antigen. After the transfer step, all three methods follow the same basic methodology for detecting the target antigen.
In the technique referred to as immunoprecipitation-Western (IP-Western), immunoprecipitation is followed by Western blotting to increase the sensitivity of detection. Throughout this manual, examples are presented of analytical reactions that are performed prior to or following the separation of proteins by SDS-PAGE. An antibody directed against one protein antigen in a complex mixture is used to identify and isolate the specific antigen. This over-simplification understates the power of immunoaffinity recognition. Within the context of this manual, immunoprecipitation is an antibody-based method of identifying and purifying a target molecule. It is possible to isolate by immunoprecipitation or immunoabsorbent procedures almost any cellular protein to which specific antibodies can be raised, provided that the molecule retains its antigenicity in the extraction system employed. In combination with newer and more sensitive analytical methods, the structure and functional associations with other molecules can be defined for a given protein or class of proteins. Immunoprecipitation is based on the ability of antibodies that specifically recognize the target antigen to form antigen-antibody complexes which can then be easily collected by capturing the complex onto a solid-phase matrix. Protein A which has a high affinity for the Fc portion of most Ig molecules, coupled to Sepharose or agarose, is usually used for this purpose. Specific binding of immune complexes to protein A is extremely rapid, occurring within seconds, and has the added feature of being extremely stable in a variety of solvent systems, whereas nonspecific binding of other proteins is low. Due to the macroscopic size of the bead, the complexes are easily collected by centrifugation. Unbound proteins are removed by choosing a wash buffer that will keep the immune-complex intact while removing all macromolecules that are not specifically recognized by the antibody. When used with radiolabeling and SDS-PAGE, immunoprecipitation can reveal important characteristics of the immuno-affinity purified target protein. Assays can be performed to determine the presence and quantity of the target protein, its apparent molecular weight, its rate of synthesis and degradation, the presence of posttranslational modifications, and if it is interacting with other proteins, nucleic acids or other ligands. These characteristics are difficult to determine using other techniques. Suitable controls are indispensable for interpreting the results. Preimmune serum or a nonrelevant antibody from the same species are frequently used to demonstrate that the Ab is specifically forming a complex with the target protein. The protein A molecule, a 42 kDa cell wall component produced by several strains of Staphylococcus aureus, contains four high-affinity binding sites capable of interacting with the Fc region of IgG from many species. The typical binding capacity of protein an immobilized to sepharose is 10-15 mg of human IgG per ml of gel.
An alternative to protein A is Protein G, a cell wall protein isolated from group G streptococci. Protein G is reported to bind with greater affinity to most mammalian immunoglobulins than protein A, although there are several species to which protein A has a greater affinity. Because the antibodies bind to proteins A and G through their Fc region, the antigen binding sites of the antibodies remain available for antigen binding. Many analytical reactions are conveniently performed on the target protein as it exists in the immune complex, prior to SDS-PAGE.
This protocol is often used following metabolic labeling and the preparation of a total cell lysate.
Protein A-Sepharose suspension
Microcentrifuge and microcentrifuge tubes
Tube rocker: Rockers, shakers, and gyrorotators are fine
2 x SDS-PAGE sample buffer
Normal Rabbit Ig
- Preclear cell Lysates to remove sticky proteins that could be carried through the procedure by adding 1-5 mI of a non-specific antibody of the type to be used to immunoprecipitate the antigen of interest (normal rabbit Ig if your antibody is made in rabbit). Add 100 mI of the protein A-Sepharose suspension and incubate at 4°C on a rocker for at least 30 min. For a minimum preclear, use a suspension of protein A-Sepharose. This step can also extend overnight. A 10% suspension of fixed protein A-bearing S. aureus Cowan I is a less expensive alternative than protein A-Sepharose for the pre-clearing step.
- Centrifuge the suspension in a microfuge for 2 min and transfer the supernatant to a new tube. Discard pelleted material as radioactive waste if using radiolabeled Lysates.
- Add an excess of the specific antibody, (usually 1-5 mI) which can be monoclonal or polyclonal, in the form of serum, ascites, culture media, or purified antibody. Add 100 mI of the protein A-Sepharose suspension and incubate at 4°C with constant shaking for at least 1 h. If necessary, add a second bridging antibody. If the specific antibody is a mouse monoclonal or rat monoclonal which has a very low affinity for protein A, a second antibody, which will serve as a bridge should be added, which will recognize the first antibody and has a high affinity for protein A. For example, if the specific antibody is a rat monoclonal IgG2a or IgG2b, add 2 IJ.I of a rabbit anti-rat antibody which will act as a bridging reagent between the specific antibody-antigen complex and the protein A-Sepharose.
- Centrifuge at maximum speed for 2 min in a microfuge and carefully aspirate off the supernatant and if radioactive dispose of it according to your institution’s guidelines. Alternatively, this material can also be used to react with another antibody.
- Resuspend the beads in 1 ml of wash buffer and repeat this step a total of five times. Choosing a wash buffer is similar to choosing a lysis buffer. The complex can be washed with the lysis buffer. Alternatively, the following stringent wash buffer can be used:50mM Tris-HCI pH 8.0,0.5 M NaCl, 5 mM EDTA, 0.02% NaN3, 0.5% Triton X-lOO, 0.5% DOC, 0.1 % SDS.
- After the final wash, add 50 ml of 2 x SDS-PAG E sample buffer directly to the beads, vortex mix, and boil for 5 min. The immunoprecipitated material can be stored at -20°C or loaded directly onto an SDS gel. Prior to loading, briefly centrifuge the tube for 20 sec and load all of the supernatant above the pelleted beads.
- Following electrophoresis, if the isotope is 35S, 14C, or 3H, treat the gel with a fluorographic enhancing agent. Dry the gel, expose it to X-ray film and analyze the data.
In parallel, include a control in which the lysate is mock immunoprecipitated with an irrelevant antibody or preimmune antisera. When electrophoresed in parallel with the immunoprecipitated material, nonspecific bands are readily identified, greatly simplifying the analysis.
Washing is important for obtaining a clean result. If you have a high background, try using a higher stringency wash buffer. If you do not detect the target protein, try decreasing the stringency of the wash buffer. You may also find that 5 washes are too many and you can achieve a low background with only 3 or 4 washes.
Sequential Immunoprecipitation – Dissociation and Reimmunoprecipitation of Immune Complexes
Cell surface receptors are often intimately associated with additional proteins which comprise a biologically active complex. It is possible to identify proteins that specifically interact with the target Ag and bind to it and are recognized as part of the immune complex although not through an affinity with the antibody. These proteins coprecipitate with the immune complex by way of a noncovalent interaction with the target protein. If you suspect that associated proteins are coprecipitating with your target protein, the complex can be disrupted and reimmuno-precipitated with a second antibody, if available.
Solution A: 50 mM Tris-HCI, pH 7.5, 137 mM NaCI
Solution B: Solution A plus 0.75% SDS, 2% 2-ME, 100 mM DTT, 10 mg/ml aprotinin, and 10 mg/ml leupeptin
Solution C: 50 mM Tris-HCl, pH 7.5,0.1 % Triton X-lOO, 137 mM NaCI 4 X and 2 x SDS-PAGE sample buffer
Protein A Sepharose suspension: Appendix C
- Perform the final immunoprecipitation wash with 1 ml of Solution A.
- Resuspend the beads in 100 ml of solution B and boil for 5 min.
- Dilute the eluted proteins tenfold with solution C.
- Remove 75 ml and mix with 25 ml of 4 x SDS-PAGE sample buffer and boil for 5 min. Analyze by SDS-PAGE followed by fluorography. This should contain the target protein and any other proteins coprecipitating with the immune complex.
- Incubate the remaining sample with a second antibody on ice for 60-90 min and then add 100 mI of the protein A suspension and incubate for an additional 1 h at 4°C.
- Wash the immune complexes three times with solution C. Add 50 mI of 2 x SDS-PAGE sample buffer and boil for 5 min.
- Analyze the precipitated products by SDS-PAGE followed by fluoro graphically enhancing the signal in the gel, drying and exposing to X-ray film.
PROTOCOL : Nondenaturing Immunoprecipitation
Another approach to test for the presence of associated proteins is to perform the cell lysis and immunoprecipitation protocol in a nondenaturing buffer system. The mild, nondenaturing conditions keep the protein complexes intact. Perform the preclearing, immunoprecipitation, and washes in the lysis buffer. All manipulations should be performed at 4°C.
Iodoacetamide solution: 0.5 M iodoacetamide in PBS
Nondenaturing lysis buffer: 50 mM HEPES, pH 7.5, 200 mM NaCI containing 2% sodium cholate, 1 mM PMSF, 5 mg/ml each of aprotinin and leupeptin
Lysis buffer: 50 mM HEPES, pH 7.5,200 mM NaCL the anti protease cocktail and 0.1 % SDS and 1 % Triton X-lOO.
Protein A-Sepharose suspension
- After labeling, incubate the cells in iodoacetamide solution for 10 min on ice.
- Lyse the cells in nondenaturing lysis buffer.
- Perform immunoprecipitation
Nondenaturing immunoprecipitation is usually followed by a denaturing immunoprecipitation. The beads are incubated with nondenaturing lysis buffer containing in addition 1 % SDS and heated at 90°C for 3 min followed by the addition of 2 ml of nondenaturing lysis buffer containing 1 % Triton X-lOO. This immunoprecipitated material is now used in the second, denaturing immunoprecipitation. Do not preclear. Add the desired specific antibody and proceed with the immunoprecipitation protocol. This technique is also referred to as sequential immunoprecipitation.