Proteins using immobilized

Guillonneau, F., Labas, V., Auvin, C., and Praseuth, D. (2001) A reliable and simple method for two-dimensional electrophoresis and identification of HeLa nuclear alkaline nucleic acid-binding proteins using immobilized pH gradient. Electrophoresis 22, 4391-4403. 

Yenugopal Y, Alur MD, Nerkar DP. Solubilization of fish proteins using immobilized microbial cells. Biotechnol Bioeng 1989 33 1098-1103. 

Gorg, A., and Weiss, W. (1998). High-resolution two-dimensional electrophoresis of proteins using immobilized pH gradients. In Cell Biology A Laboratory Handbook (J. E. Celis, ed.), 2nd ed., Vol. 4, pp. 386-397. Academic Press, San Diego, CA. 

Molloy, M.P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal. Biochem. 280, 1-10. 

High-Resolution Two-Dimensional Electrophoresis of Proteins Using Immobilized pH Gradients... 

Liver is 1 of the tissues most frequently analyzed for contaminant residne in wildlife, but maybe 1 of the least useful because of the poor correlation between fiver mercniy concentration and effects, and because of the tendency of the liver to accumulate mercury over time (Stewart et al. 1999 Scheuhammer et al. 2001). Liver is a major site of demethylation therefore, the proportion of fiver mercury present as MeHg is not representative of exposure to MeHg. Moreover, most mercury in fiver is botmd to metallothionein or other suUydryl-bearing proteins, which immobilize it (Med-insky and Klaassen 1996 Yasutake etal. 1997 Aschner 1999). Therefore, fiver mercury residue values must be used with caution, and only when more suitable tissues are unavailable. 

Wu, H. and Bruley, D.F, Homologous Human Blood Protein Separation Using Immobilized Metal Affinity Chromatography Protein C Separation from Prothrombin with Application to the Separation of Factor IX and Prothrombin, Biotechnol. Prog., 15, 928, 1999. 

A variety of approaches exist for stabilizing proteins, for example, chemical modification, immobilization, and site-directed mutagenesis [95,96], but these techniques are not within the scope of this chapter. The focus here will be on stabilization of proteins via formulation development. The principal formulation strategy is to stabilize the protein using clinically acceptable additives (excipients) or through the use of suitable pharmaceutical-processing technologies. 

Norbeck J et al. Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension reproducibility and evidence for isoelectric focusing of alkaline (pi >7) proteins. Yeast 1997 13 1519-1534. 

COOH groups of the PE (PAA or PGA) and the -NH2 moieties of the pre-loaded protein, using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a catalyst [99]. Negligible enzyme desorption (<0.1 %) is observed from cross-linked lysozyme-loaded MS spheres after exposing the samples to an aqueous solution for 48 h, while about 25 % of the immobilized lysozyme is desorbed under the same conditions when the lysozyme is not cross-linked. 

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins, using a carbodiimide reaction with EDC. A similar protocol to that previously described for coupling to carboxylate polymer particles may be used. The following protocol is based on the method of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli 0157. 

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Recover interaction complexes by affinity chromatography. This is typically done using immobilized antibodies to the bait protein or using fusion tag binders, if the bait protein contains a fusion partner (e.g., immobilized glutathione for binding GST labeled bait proteins). 

---