The final supernatant is the pre-cleared S100 fraction. Spin the pre-cleared S100 at 35,000rpm in Beckman type Ti70.1 rotor for 15 min at 4C. Carefully collect the supernatant, and transfer to a 10ml disposable syringe attached to a glass fiber pre-filter disc (Millex-AP). Filter the S100 fraction through the glass fiber pre-filter disc into a new 15ml conical bottom tube to capture contaminating lipids and minimize carryover of beads from the pre-clearing steps. Proceed to immunoaffinity purification using the procedures described in section 3.3.2, steps 4 to 26. 3.3.4. conventional chromatography-based approaches have long been used for protein purification, they suffer from a number of disadvantages. First, many multi-protein complexes are quite fragile and are not stable to the extremes of ionic strength or other conditions encountered during ion exchange, hydrophobic interaction, gel filtration, or other forms of conventional chromatography. Second, the degree of purification that can be obtained using any one separation method is typically limited, and it is almost always necessary to develop time-consuming and technically challenging strategies that combine multiple purification steps. The use of immunoaffinity purification strategies can alleviate many of the problems associated with conventional chromatography. In an immunoaffinity purification, an antibody that recognizes a protein of interest is bound to a resin such as agarose or Sepharose beads. A cell extract or partially purified fraction is passed over the antibody-resin, unbound proteins are washed away, and specifically bound proteins are then eluted from the antibody with competing epitope peptides or by more harsh treatments that result in complex dissociation or loss of activity, such as high salt or brief exposure to acidic pH. Using such methods, it is possible to achieve substantial purification in a single step; however, successful application of immunoaffinity approaches is dependent on the availability of antibodies with suitable affinity and specificity. It is often not possible to obtain antibodies suitable for Trimethobenzamide hydrochloride immunoaffinity purification for each individual protein that one wishes to study. An alternate strategy takes advantage of well-characterized antibodies that recognize short, defined peptide sequences with high specificity and affinity. These sequences, referred to as epitope tags, are added to either the amino- or carboxyl-terminus of a protein of interest (1). When expressed in mammalian cells, the epitope tagged protein can be incorporated into a protein complex or complexes in place of its endogenous counterpart, allowing purification of the tagged protein and any proteins with which it is associated by immunoaffinity chromatography using anti-epitope antibodies (See Note 1). Table 1 shows a list of commonly used epitope tags for immunoaffinity purification (2C5). Table 1 Useful Epitope Tags and Resins for Immunoaffinity Purification thead th valign=”top” align=”left” rowspan=”1″ colspan=”1″ Tag /th th valign=”top” align=”left” rowspan=”1″ colspan=”1″ Epitope peptide sequence /th th valign=”top” align=”left” rowspan=”1″ colspan=”1″ CACNB4 Affinity resin /th th valign=”top” align=”left” rowspan=”1″ colspan=”1″ Binding specificity /th /thead FLAGDYKDDDDK-FLAG M2 agarose (SIGMA)N, Met-N, Internal, CHAYPYDVPDYA-HA agarose (HA-7, SIGMA)N, C-HA agarose (HA.11, Covance)N, Internal, CcMycEQKLISEEDL-cMyc pAb agarose (SIGMA)N, C-cMyc agarose (9E11, Santa cruz)N, CV5GKPIPNPLLGLDST-V5 agarose (V5-10, SIGMA)N, C Open in a separate window Elution from antibody affinity resins is typically performed using peptides composed of 1 or 3 consecutive repeats of the epitope sequence. A general strategy for the use of epitope-tagging and immunoaffinity purification of protein complexes is outlined in Figure 1. The first step is to construct a suitable expression vector that encodes an epitope tagged protein that can be expressed in mammalian cells. The second step is to generate and amplify clonal cells stably expressing useful amounts of the epitope tagged protein. Finally, Trimethobenzamide hydrochloride the protein of interest and any associated proteins can be purified from nuclear or cytoplasmic extracts by single-step immunoaffinity purification by binding to immobilized anti-epitope antibody and competitive elution with epitope peptides. Using this approach, we have successfully used anti-FLAG epitope immunoaffinity purification to purify the human Mediator of RNA Trimethobenzamide hydrochloride polymerase II to near homogeneity from extracts of HeLa S3 cells stably expressing any of a large number of FLAG-epitope tagged Mediator subunits (6) (Figure 2). Notably, using cell lines expressing FLAG-tagged versions of mutant Mediator subunits, we have been able to purify mutant Mediator complexes that have proven useful in functional studies (7). Open in a separate window Figure 1 Scheme For Immunoaffinity Purification of Protein Complexes Open in a separate window Figure 2 Immunoaffinity Purified Mammalian Mediator Complex From HeLa S3 Nuclear Extract Through FLAG-tagged Mediator Subunits. 2. Materials 2.1 Production of Mammalian Cell Lines Host cells (e.g. HeLa S3 cells, HEK293/FRT cells) Expression vector encoding epitope-tagged protein of interest Antibiotic needed for drug selection of stably transformed cells 2.2 Cell Extract Preparation 0.4% (w/v) Trypan Blue Solution in PBS (#25-900-Cl, Mediatech) Hypotonic buffer (10 mM HEPES (pH7.9), 1.5mM MgCl2, 10 mM KCl, 0.5 mM Dithiothreitol) Extraction buffer (20 mM HEPES (pH7.9), 1.5 mM MgCl2, 0.6 M KCl, 0.2 mM EDTA, 0.5 mM Dithiothreitol, 25% Glycerol) Phosphate buffered saline Buffer C (20 mM HEPES (pH7.9), 1.5mM MgCl2, 0.2 mM EDTA, 0.5 mM Dithiothreitol, 25% Glycerol) Protease inhibitor (P8340, SIGMA), add to all buffers immediately before use Dounce homogenizer (40 ml, 15 ml, and/or 7 Trimethobenzamide hydrochloride ml) 15 and 50.