Electrophoresis band migration

PGE was isolated as desribed in Material and Methods. SDS-PAGE electrophoresis of purified protein showed a single band migrating at approximately 60 kDa. This observation is not in the agreement with the calculated molecular weight of 35 584. However a similar effect has been observed previously in case of PGI and PGC. Apart of the N-glycosylation which plays role in all PGs (Fig. 3), O-glycosylation may also be present as indicated by the band size shift after a treatment of PGE with O.IM NaOH (data not shown). 

Isotachophoresis. In isotachophoresis (ITP), or displacement electrophoresis or multizonal electrophoresis, the sample is inserted between two different buffers (electrolytes) without electroosmotic flow. The electrolytes are chosen so that one (the leading electrolyte) has a higher mobility and the other (the trailing electrolyte) has a lower mobility than the sample ions. An electric field is applied and the ions start to migrate towards the anode (anions) or cathode (cations). The ions separate into zones (bands) determined by their mobilities, after which each band migrates at a steady-state velocity and steady-state stacking of bands is achieved. Note that in ITP, unlike ZE, there is no electroosmotic flow and cations and anions cannot be separated simultaneously. Reference 26 provides a recent example of capillary isotachophoresis/zone electrophoresis coupled with nanoflow ESI-MS. 

HDL, respectively. By starch block, some of the VLDL exhibit 0 2-mobility, whereas by polyacrylamide gel electrophoresis the pre-j8 band migrates in post-)8 position, a phenomenon due to the sieving effect of the supporting medium. 

On starch gel electrophoresis, gastricsin migrated 6.5 cm toward the anode after 22 hours as one single narrow band in acetate buffer of pH 5.0. These two materials also differed in heat sensitivity while pepsin at pH 2.0 and 65° C lost 69% of its activity, gastricsin under these conditions lost 44.8%. Conversely, at pH 3.2 pepsin was inactivated only 11.2%, while gastricsin was inactivated 22.3% (Fig. 4). Both enzymes hydrolyzed synthetic carbobenzoxy-glutamyl-L-tyrosine, which is a specific substrate for pepsin. Gastricsin was crystallized as it came from... 

Electrophoresis The buffer reservoirs are filled with cold (about 5°C) electrode buffer and any bubbles trapped at the top or bottom of the gel are removed. Electrophoresis is carried out at a constant current of 4 mA/tube (applied voltage about 90 V at the outset). Electrophoresis is continued until the bromophenol blue band migrates to about 1 cm from the bottom of the tube, usually about 70 min. 

TTR migrates anodal to albumin on routine serum electrophoresis the presence of a TTR band is considered one marker of good quality electrophoresis. However, levels can be only roughly semiquantified from the intensity of the band. RBP dissociates during electrophoresis and migrates anodal to transferrin, but the band is usually too faint to see. Either protein can be quantified by routine immunochemical methods. 

HPLC analysis of a cordocentesis blood sample shows one or two. very sharp and narrow peaks at the injection point on the chromatogram (Figure 31-12, a). The major band is Hb Bart s with a smaller band attributed to Hb Portland. There is a complete absence of Hb E Alkaline electrophoresis shows a band migrating at or close to the solvent front (Hb Bart s) with another band in the Hb A position (Hb Portland). 

In electrophoresis, band broadening is mainly caused by longitudinal diffusion. The peak dispersion, o, is directly proportional to the diffusion coefficient, D, of the analyte and its migration time, t ... 

Are we truly in the low-field limit Most electrophoretic studies emphasize circumstances that permit separations. Electrophoresis at very low field, with band broadening overwhelming band migration, will not give separations, but from the above may be interesting for polymer physics studies. 

Disc Electrophoresis. Resolution in zone electrophoresis depends critically on getting sample components to migrate in a focused band, thus some techniques ate employed to concentrate the sample as it migrates through the gel. The most common technique is referred to as discontinuous pH or disc electrophoresis. Disc electrophoresis employs a two-gel system, where the properties of the two gels are different. 

Figure 7.1 Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulfide linkages). The electric field is applied across the gel after the protein samples to be analysed are loaded into the gel wells. The rate of protein migration towards the anode is dependent upon protein size. After electrophoresis is complete, individual protein bands may be visualized by staining with a protein-binding dye (a). If one well is loaded with a mixture of proteins, each of known molecular mass, a standard curve relating distance migrated to molecular mass can be constructed (b). This allows estimation of the molecular mass of the purified protein...

Figure 8.5 Effect of pH on protein mobility. Hemoglobin A (pi 7.1) and Hemoglobin C (pi 7.4) were electrophoresed in eight of the McLellan native, continuous buffer systems (Table 8.1). The diagram is drawn to scale. Migration is from top to bottom as shown by the vertical arrows. Bands marked A or C indicate the positions of the two hemoglobin variants in each gel representation. The polarities of the voltages applied to the electrophoresis cell are indicated by + and - signs above and below the vertical arrows. Run times are shown below the arrows. Note the polarity change between the gel at pH 7.4 and the one at pH 8.2. This reflects the pis of the two proteins (and was accomplished by reversing the leads of the electrophoresis cell at the power supply).

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