Association membrane sampling technique

The membrane sampling technique was used extensively to get samples which were representative of the solution in equilibrium with the associated complexes. Briefly, the sample is equilibriated in a sealed polyethylene container under nitrogen at least overnight. A 200 mL portion of the sample is placed in the ultrafilter cell (Amicon 8200) with an XM300 membrane (Amicon 300,000 MWCO). Earlier studies (Woerner, D. L. McCarthy, J. 

No major differences in polypeptide composition or staining intensity between the two samples are apparent. For both cases, membranes were UV irradiated in the presence of azido- C-atrazine prior to electrophoresis. Analysis of the gel by a fluorographic technique showed no detectable bound radiolabel associated with the membrane sample from resistant chloroplasts. 

Another technical challenge using the 2-DE-based approach relates to its narrow dynamic range of protein detection, a limitation that makes it almost impossible to detect and compare the expression of many very low abundance proteins, even with sample subfractionation. Though various aforementioned protein enrichment techniques are available, the application of LC-based protein separation coupled with highly sensitive mass spectrometric protein identification can address the issue of dynamic range, to a certain extent. This approach also improves the resolution and identification of proteins with extreme pi, large mass, and, in some cases, membrane associations. 

As previously discussed, electron, light, and confocal microscopy techniques may be used to visualize the position of electron-dense precipitates, radioactive substances, and fluorescent probes, respectively, in the sample tissue. However, none of these techniques possess the capability both to visualize and to selectively measure the flux of a molecule across the skin. SECM, however, permits the measurement and subsequent imaging of the local flux of an electroactive species across biological membranes. Scott et al. [3] used SECM to investigate the effect of pretreatment of the penetration enhancer sodium dodecyl sulfate (SDS), on the ion transport rate and transport pathways of Fe(CN) across hairless mouse skin. Increasing the time of SDS exposure from 10 min to 30 min increased the overall (porous and nonporous) transport of Fe(CN) by 17-fold. More specifically, the SDS-induced increase in Fe(CN)g transport was found to be associated with nonporous (i.e., intercellular) transport routes, while transport via porous routes was significantly reduced. The fraction of Fe(CN)g transport through pores, as measured by... 

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