Affinity polyacrylamide

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.

Cuatrecasas, P. (1970). Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J. Biol. Chem. 245, 3059-3065. 

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

According to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and CaM affinity chromatography analyses, compounds 44—53 interacted with both spinach and bovine-brain CaMs. In order to quantify the interaction of the phytotoxic agents 44—53 with... 

Fig. 2. Alternation ot gel-shitt and filter-binding selection steps Target-bound and unbound radiolabeled RNA aptamers are separated by polyacrylamide gel electrophoresis, visualized by autoradiography, purified from the gel, and used for the subsequent nitrocellulose-filter binding selection step. The experiments are earned out in the presence (-i-) and absence (-) of target protein using the SELEX cycles 0 (control), 3, and 7. The figure illustrates the increase of binding affinity of selected RNA pools, seen as augmented quantity of RNA retained together with the receptor protein at the top of the gel (modified from ref. (8)).

Polj/meric supports based on polyacrylamide are s)mthesized by copol3tmerization of acrylamide and a cross-linking reagent and can be used directly in affinity chromatography due to its more hydrophilic properties than polystyrene supports. Polyacrylamide gels are either soft... 

Analysis by SDS-polyacrylamide gel electrophoresis of purified NOS from isolated HC from rats injected with killed Corynebacterium parvum (H), and from the murine macrophage cell line RAW 264-7 (M) (courtesy of D. Stuehr, The Cleveland Clinic, Cleveland, OH) which was exposed to LPS and IFNy. Crude cytosols were separated using ion exchange and affinity 2 5 -ADP Sepharose chromatography. Last step by gel filtration is equivalent to separation by molecular weight. 

Thus one might expect flexible, water-soluble synthetic polymers with suitable side chains to show strong affinities for small molecules. In the course of 20 years we examined the binding ability of polyvinylpyrrolidone, polyvinylpyridine, polylysine, polyacrylamide, polyisopro-pylacrylamide, polyvinylimidazole, polyvinylmethyloxazolidinone,... 

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