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New solution IEF device for micropreparative separation of peptides and proteins
Author(s) -
Duša Filip,
Šlais Karel
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200485
Subject(s) - chromatography , chemistry , fractionation , separation process , fraction (chemistry) , ethylene glycol , organic chemistry
The article presents a new concept of preparative solution IEF where time requirements and efficiency are similar to gel‐based IEF whereas simple fraction handling as well as quick and complete protein recovery typical for solution‐based IEF methods are maintained. The presented method is based on the IEF in separation medium soaked in a segmented strip of nonwoven fabric. The strip is positioned in an open horizontal V ‐shaped trough. Suggested focusing method combines free solution IEF under continuous evaporation and whole channel dispensing. Separation medium based on ethylene glycol/water mixture enhances viscosity enough to reduce electroosmosis and prevents the medium from completely drying out. Generation of p H gradient and final local p H is visually traced by colored low‐molecular p I markers added to input mixture, which enables an optimization of focusing process and collection of individual fractions at desired p H range. The proposed method was tested by fractionation of the proteins and bioactive peptides originating from raw whey. Moreover, subsequent HPLC analysis of the individually collected solution IEF fractions was used for identification of whey components. We confirmed that the method is capable to process directly few tenths of milliliters of raw samples including the salty ones.