Purification affinity

Nucleic acids such as RNA and DNA can be separated from a solution using a complementary probe. For example, the strong affinity between 

Wash and remove supernatant, add eluent, resuspend and immobilize beads with magnet 

A novel approach to specific binding involves the use of triple-helix affinity capture [26,27]. Triple-helix DNA has proven to be a useful approach to 

The primary objective of a chromatographic screening purification is to obtain sufficient purified protein for preliminary experiments. Affinity chromatography is the method of choice in this type of research, avoiding complex method development of multi-stage purification protocols. 

After sample preparation, such as cell disruption, centrifugation, and/or filtration, a small amoimt of solution (a few mL) is applied to a small column. 

It is advisable to have some ready-to-use commercially available affinity columns to hand, such as IMAC resins, p-aminobenzamidine. Reactive Blue, heparin. Protein A for antibody purification, as well as some activated resins such as tresyl, epoxy or N-hydroxysuccinimide. 

Most resins can be stored for more than two years, but activated resins are hydrolytically labile and should be used immediately upon opening the container [63]. 

Usually, an affinity purification consists of the following steps  

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Manenti S, Sorokine O, Van Dorsseiaer A and Taniguchi H 1992 Affinity purification and characterization of myristoyiated aianine-rich protein kinase C substrate (MARCKS) from bovine brain J. Biol. Chem. 267 22 310-15... 

Cells contain thousands of different proteins. A major problem for protein chemists is to purify a chosen protein so that they can study its specific properties in the absence of other proteins. Proteins have been separated and purified on the basis of their two prominent physical properties size and electrical charge. A more direct approach is to employ affinity purification strategies that take advantage of the biological function or similar specific recognition properties of a protein (see Table 5.5 and Chapter Appendix). 

Narum, D. L., Welling, G. W., and Thomas, A. W., Ion-exchange-immuno-affinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1, /. Chromatogr. A, 657, 357, 1993. 

Figure 2.2. Fractionation of protein extracts before 2D gel electrophoresis. Crude lysates can be fractionated by affinity purification or by a number of chromatographic techniques. In addition, organelles or other cellular structures can be purified and lysates from these organelles can be fractionated or separated directly on 2D gels. By repeating this procedure using a number of conditions it may be possible to visualize a large fraction of a cell s proteome.

The endothelin B receptor is an example of characterization of a homogeneous, affinity purified protein (Roos et al., 1998). Significant progress has been made in the development of techniques for more high-throughput identification of phosphorlyation events. Analysis of large sets of phosphorylated proteins is facilitated by the availability of affinity purification methods such as anti-phosphotyrosine or anti-phosphoserine antibodies or metal affinity chromatography (Neubauer and Mann, 1999 Soskic et al., 1999). These methods are not specific to a particular protein but rather are used to fractionate all proteins that are phosphorylated. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

We have previously shown that a 209 amino acid region (aa288-497, asymmetric localization domain) of Insc is necessary and sufficient for apical cortical localization and for mitotic spindle orientation along the apical-basal axis (Tio et al 1999). In a yeast two-hybrid screen we identified Partner of Inscuteable (Pins), a novel 658aa protein with multiple repeats of the Tetratricopeptide (TPR) motif. Affinity purification experiments using embryonic extracts demonstrate that Pins complexes with Insc in vivo. In vitro protein interaction assays demonstrates that Pins interacts with the Insc asymmetric localization domain (see Yu et al 2000). 

Graumann, J., Dunipace, L.A., Seol, J.H., McDonald, W.H., Yates, J.R., 3rd Wold, B.J., Deshaies, RJ. (2004). Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast. Mol. Cell. Proteomics 3, 226-237. 

Batch Ni2+ affinity purification of elF3 (Ni pull-down [Ni PD]) 23... 

It should be emphasized that the nature of all presented protocols is very general and, thus, their application for a comprehensive characterization of your favorite multiprotein complex (YFMPC) in yeast might require only minor modifications. The logical sequence of all required steps is schematically shown in Fig. 2.1. The initial large-scale Ni affinity isolation of eIF3 followed by mass spectrometry (MS) of its subunit composition has already been described (Asano et al, 2002), and methods for identification of protein-protein interactions such as yeast two-hybrid (Y2H) and in vitro glutathione-S-transferase (GST) pull-down analysis are presented in volume 429. This chapter focuses on a description of the small-scale one-step in vivo affinity purification techniques that were used to determine the effects of deletions and... 

Deletion/Mutational Analysis of eIF3 Subunits and Ni2+ Affinity Purification of Their Subcomplexes... 

We have employed a more systematic random mutagenesis approach by dividing the BDI into consecutive clusters of 10 amino acid residues (Boxes) that are individually replaced with a string of 10 alanine residues in the full-length Hisg-tagged allele to facilitate affinity purification of the mutant protein (Valasek et ah, 2004). Alternatively, clusters rich in... 

Edery, I., Altmann, M., and Sonenberg, N. (1988). High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin. Gene 74, 517-525. 

Aliquot 2 /A DMSO into sample 1 (carrier control) and sample 2 (affinity purification), and 2 fiM 2 mM PatA into sample 3 (competition), followed by incubation at 4° with mixing for 1 h. 

Recently, Vasilescu et al. demonstrated the use of formaldehyde to preserve protein interactions in vivo followed by immunoaffinity purification of a targeted complex, cross-link reversal via heating at 95°C, separation by SDS-PAGE, and identification of bands by LC-MS/MS.7 Tagwerker et al. utilized formaldehyde cross-linking in conjunction with a novel tag-based affinity purification method.36... 

Puig O, Caspary F, Rigaut G, et al. The tandem affinity purification (TAP) method a general procedure of protein complex purification. Methods 2001 24 218-229. 

Aithal, H.N., Knigge, K.M., Kartha, S., Czyewski, E.A., and Toback, F.G. (1988) An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies. /. Immunol. Meth. 112, 63-70. 

Zopf, D.A., Smith, D.F., Drzeniek, Z., Tsai, C.-M., and Ginsburg, V. (1978a) Affinity purification of antibodies using oligosaccharide-phenethylamine derivatives coupled to Sepharose. In Methods in Enzymology, (V. Ginsburg, ed.), Vol. 50, pp. 171-175. Academic Press, New York. 

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

Sample Clean-Up through Affinity Purification Employing Molecularly... 

Expression of a recombinant protein using an inducible vector system would permit expression at endogenous levels to simulate physiologic levels of expression of a protein of interest. Tandem affinity purification strategies have recently been employed and facilitate the analyses of highly interactive proteins when the bait protein is expressed at endogenous levels. Immunoaffinity or immunoprecipitation followed by LC-MS/MS does not readily permit determination of the stoichiometry of interacting partners. Additionally, when compared to yeast hybrid experiments, it is difficult to determine whether interactions are binary when identified in complexes by MS/MS. 

Pitt JC, Lindemeier J, Habbes HW, Veh RW (1998) Haptenylation of antibodies during affinity purification a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling. Histochem Cell Biol 110 311 322 Polak JM, Van Noorden S (1997) Introduction to immunocytochemistry. BIOS Scientific... 

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