A horizontal apparatus for isoelectric protein purification in a segmented immobilized pH gradient.
A modification of the previously described apparatus (Faupel et al. (1987) J. Biochem. Biophys. Methods 15, 147-162), for recycling isoelectric focusing in a segmented immobilized pH gradient, is here reported. The most important improvements are: (1) a horizontal, vs. the previously vertical assembly; (2) a reduction of the thickness of the central flow chamber to 6 mm, vs. the previous 3 cm length and (3) the introduction, at both gel extremities of each Immobiline segment, of polypropylene filters, thus efficiently blocking the gel in situ. The advantages are: (i) the spontaneous removal of air bubbles, which in the vertical apparatus tend to accumulate in the ceiling of the flow chamber and to obstruct the flow of electric current; (ii) a more efficient hydraulic flow with a reduced chance of heating the liquid stream in the flow chamber, due to its reduced length along the separation path and (iii) a reduced risk of gel detachment from the tube walls, due to osmotic swelling caused by focused protein zones in the gel phase and by the fixed Immobiline charges in the polyacrylamide matrix.
Righetti
P G
PG
Department of Biomedical Sciences and Technologies, University of Milan, Italy.
Barzaghi
B
B
Luzzana
M
M
Manfredi
G
G
Faupel
M
M
D000328:Adult; D004867:Equipment Design; D006454:Hemoglobins / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation*; D011506:Proteins / Q000302:isolation & purification*
D006454:Hemoglobins; D011506:Proteins