In protein extraction

Sample preparation used to extract proteins from cells prior to analysis is an important step that can have an effect on the accuracy and reproducibility of the results. Proteins isolated from bacterial cells will have co-extracted contaminants such as lipids, polysaccharides, and nucleic acids. In addition various organic salts, buffers, detergents, surfactants, and preservatives may have been added to aid in protein extraction or to retain enzymatic or biological activity of the proteins. The presence of these extraneous materials can significantly impede or affect the reproducibility of analysis if they are not removed prior to analysis. 

Unlu, M., Morgan, M.E., Minden, J.S. (1997). Difference gel electrophoresis a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071-2077. 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

The first data confirming this oxidoreductive epimerization were obtained by measuring the enzyme activity in protein extracts from a tomato cell suspension [24], It could be demonstrated, using a very sensitive fluorimetric detection method, that two different enzymes were involved in this subpathway. No enzyme activity could be detected with 24-ep/-teasterone as substrate and NAD+ or NADP+ as electronacceptors. But using the proposed intermediate 3-dehydro-24-epi-teasterone as substrate, enzymatic conversion to 24-epi-teasterone was measured in a microsomal fraction of tomato cell cultures (Fig. (9)). 3-dehydro-24-epi-teasterone-reductase showed a specific activity of 361 fkat/mg protein with NADPH as the only accepted electrondonor. 

Enzyme activities were detectable only in protein extracts derived from cell cultures after induction with the corresponding substrates. Two different enzyme activities were shown to be involved in this subpathway, one located in the microsomal fraction, and one with a typical cytosolic dehydrogenase activity. 

On the other hand, heat inducibility of these gene constructs (Fig. L4) seems to be drastically impaired in imbibed seeds. Seeds of 5xHSE2 CaMV-CATter derived plants show a small but insignificant increase of the otherwise basal levels of CAT activity after hs (Fig. 1). However, this maximum level of CAT activity is much lower than the basal levels measured in protein extracts of leaves from the non-induced parental plants (Fig. 1). Two to 20 hours of imbibition prior to the heat treatment... 

The synthesis of BOA-6-O-glucoside is catalyzed by constitutive enzymes that may be upregulated. A glucosyltransferase that accepted BOA-6-OH as a substrate was measurable in protein extracts of corn roots harvested from control plants. Detoxification via glucoside carbamate synthesis is inducible and seems to be more complicated than simple A/-glucosylation (data unpublished). The biosynthesis of the compounds is part of our ongoing research. 

Using SELDI technology, a-defensin isoforms were found to be elevated in serum from colon cancer patients and in protein extracts from CRC [59]. This result was confirmed by expression analysis of microarray data obtained from 283 tumors and normal tissues followed by serum analysis of colon cancer patients and controls by ELISA. This study yielded a diagnostic sensitivity of 70%i and specificity of 83% for a-defensin in colon cancer [60]. Although these figures appear too low for developing a screening test, this... 

Proteins solubilized in aqueous solution interact more or less with hydrophilic groups of surfactants at the oil-water interface. Therefore, the type of hydrophilic group is strongly influenced by the protein extraction efficiency. Anionic and cationic surfactants interact with charged protein surfaces more strongly than non-ionic surfactants. This feature also means that the non-ionic surfactants are favourable for protein stabilization in water droplets because of the not-so-hard interaction between the protein and the surfactant. In protein extraction, such an electrostatic interaction between proteins and surfactants is the main driving force in protein transfer. 

The interpretation of the above results is made more difficult by a supplementary phenomenon due to the aqueous phase ionic strength. Increasing the density of charges in the vicinity of the surfactant headgroups causes a screening effect not only between surfactant and protein but also in the repulsive interaction between surfactant polar heads they come closer and the micellar size decreases (43). As a consequence, Goklen and Hatton (48) noted a decrease in the amount of water in AOT system when increasing KCl concentration. However, it is difficult from these results to state precisely whether the decrease in protein extraction is due to the decrease of electrostatic interactions or to a size exclusion effect. 

---