Preparative isoelectric focusing in a cellulose‐based separation medium

Abstract

An improved preparative method based on isoelectric focusing of analytes in a cellulose-based separation medium is described in this study. Cellulose is suspended in an aqueous solution of simple buffers, ethylene glycol, glycerol, nonionic surfactant, and colored pI markers. Water partially evaporates during focusing run and the separation takes place in an in situ generated layer of cellulose, which has a gel-like appearance at the end of analysis. Final positions of analytes are indicated by the positions of zones of focused pI markers. Fractions, segments of the separation medium with analytes, can be simply collected by spatula and analyzed by downstream analytical methods. Good focusing ability of the new method and almost quantitative recovery of model proteins, cytochrome c and bovine serum albumin, was verified by gel electrophoresis and capillary isoelectric focusing of the collected fractions.

Preparative and capillary isoelectric focusing for detection and identification of Aspergillus conidia in complex sample matrices

“…Preparative IEF (pH gradient pH 2–10.1) of conidia of A. niger (A,B(i),B(iv)), A. fumigatus (B(ii), B(iv)), A. flavus and A. parasiticus (B(iii), B(iv)); (A) 10 7 conidia mL −1 in PSS or (B) 10 6 conidia mL −1 in 0.45% (m/v, %) NaCl with 1% (v/v, %) Tween 80 and 7% (v/v, %) 1‐butanol; 100 μL sample was injected into the trough with the separation medium in the original composition , samples (A), and with optimized composition of the working suspension, samples (B); pI markers pI 2.0, 2.6, 3.9, 5.3 and 10.1; (C) the cultures of Aspergillus spp. from a collected fraction of the focused conidia in the trough after a 7‐day cultivation…”

“…2‐[4‐(2‐Hydroxyethyl)‐1‐piperazinyl]‐ethanesulphonic acid was obtained from Merck (Darmstadt, Germany) and L‐aspartic acid from LOBA Chemie (Vienna, Austria). PEG ( M w 10 000), acetone, ethanol (EtOH), 1‐butanol, α–cellulose (powder, cat No C8002, average fibre length 200 μm), ethylene glycol, glycerol, Brij 35, Tween 80, Triton X‐100, 2‐(4‐Sulfophenylazo)‐1,8‐dihydroxy‐3,6‐naphthalenedisulfonic acid trisodium salt, and individual amphoteric compounds (used as simple buffers in the separation medium ) were purchased from Sigma‐Aldrich (Schnelldorf, Germany). All chemicals were of analytical grade.…”

“…All chemicals were of analytical grade. Specifications of the simple buffers used as spacers were described elsewhere . Colored pI markers for preparative IEF‐pI 2 (green), 2.6 (orange), 3.9 (orange), 5.3 (lavender), 5.7 (orange), 6.2 (red), and 10.1 (violet) and for CIEF pI 2, 2.7, 4, 5.3, and 7 were developed and synthesized at the Institute of Analytical Chemistry of the CAS, v. v. i.…”

“…The preparative IEF device, including its photograph, was described previously . The procedure was similar to the one in . However, the composition of the IEF separation medium had to be modified for the analysis of the conidia.…”

“…The use of capillary techniques is limited by tens of nanoliters of a homogeneous sample which can be injected directly into the separation capillary. Recently used preparative method based on IEF allowed the preseparation and preconcentration of colored microbes . It was a question of whether it is possible to separate highly hydrophobic conidia by this technique from complicated sample matrices.…”