Single-input divergent flow IEF for preparative analysis of proteins.
The instrument for continuous divergent flow IEF based on our principles set and outlined previously was further extended and tested. The separation and focusing area of a trapezoidal shape had a porous bed made from a nonwoven textile material with thickness decreasing from narrow input to wide output. A narrow end was used as a single input to continuously bring a single solution into separation space with a flow rate of 0.18 mL/min. Two pairs of electrodes were positioned close to both narrow and wide ends of the separation area with equilibrium state voltage of 75 V at the narrow input and 384 V at the wide output. Under dynamic equilibrium state, the zones of both pH gradient components and analytes were separated close to the input point and focused with increasing resolution while transporting through the separation space. The long-term stability experiments had shown the suitability of the device for preparative analysis; the zones of pI markers, hemoglobin and cytochrome C remained focused and separated over 15 h with deviations from the mean focusing positions ranging from 1.26 to 3.96% of the bed output width.
Stastna
Miroslava
M
Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic.
Slais
Karel
K
D045304:Cytochromes c / Q000302:isolation & purification; D006454:Hemoglobins / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D011091:Polyesters / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D013997:Time Factors
D006454:Hemoglobins; D011091:Polyesters; D011506:Proteins; D045304:Cytochromes c