Affinity purified

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.5 presents a summary of a purification scheme for a selected protein. Note that the specific activity of the protein (the enzyme xanthine dehydrogenase) in the immuno-affinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, xanthine dehydrogenase in fraction 5 versus fraction 1 is enriched more than 1400-fold by the purification procedure. 

Nesheim M., Blackburn M. N., Lawler C. M., Mann K. G. Dependence of antithrombin III and thrombin binding stoichiometries and catalytic activity on the molecular weight of affinity purified heparin. J Biol Chem 1986 261,3214-21. 

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. 

Pool 96 yeast strains from each plate Affinity purify GST fusions in batch... 

Knox, D.P., Smith, S.K. and Smith, W.D. (1999) Immunization with an affinity purified protein extract from the adult parasite protects lambs against Haemonchus contortus. Parasite Immunology 21, 201-210. 

Verma R et al. Proteasomal proteomics identification of nucleotide-sensitive pro-teasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 2000 11 3425-3439. 

Nakamura KC, Kameda H, Koshimizu Y, et al. Production and histological application of affinity-purified antibodies to heat-denatured green fluorescent protein. J. Histochem. Cytochem. 2008 56 647-657. 

Guerrero C, Tagwerker C, Kaiser P, et al. An integrated mass spectrometry-based proteomic approach quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol. Cell. Proteomics. 2006 5 366-378. 

Hurst, G.B., Lankford, T.K., and Kennel, S.J. (2004) Mass spectrometric detection of affinity purified cross-linked peptides./. Am. Soc. Mass Spectrom. 15(6), 832-839. 

Osborn, M., and Weber, K. (1982) Immunofluorescence and immunocytochemical procedures with affinity purified antibodies Tubulin-containing structures. Meth. Cell Biol. 24, 97-132. 

Purified antibodies are usually prepared from whole serum by affinity chromatography. Concentration of affinity-purified specific antibodies is normally specified by the manufacturer in the antibody data sheet. When starting with a new... 

Affinity-purified antibodies are isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads. 

For immunoaffinity matrix, to affinity-purify the antibodies generated... 

Hul5 is a stoichiometric component of affinity-purified yeast 26S proteasomes purified under low-salt conditions [80], Hul5 is a HECT-domain E3 ligase known as KIAAIO in mammals. It assembles both K48- and K29-linked polyubiquitin chains and binds to PA700 and to isolated Rpnl/S2 via an N-terminal domain... 

Veema, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Dohmen, J., and Deshaies, R. J. Proteasomal proteomics identification of nucleotide-sensitive proteasomal-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Bid. Cell 2000, 71, 3425-3439. 

Secondary antibody affinity-purified fluorescent antiglobulin conjugate reactive with the species globulin of the first step, e.g., affinity-purified rhodamine-con-jugated goat antimouse IgG(H + L chains) (Jackson ImmunoResearch, West Grove, PA), diluted to 25 pg/mL in BSA-PBS. Can be stored at-70°C and reused (see Notes 3 and 4). 

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