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Characterisation of wool intermediate filament proteins separated by micropreparative two‐dimensional electrophoresis
Author(s) -
Herbert Ben R.,
Molloy Mark P.,
Yan Jun X.,
Gooley Andrew A.,
Bryson Warren G.,
Williams Keith L.
Publication year - 1997
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.1150180339
Subject(s) - chemistry , isoelectric focusing , chromatography , electrophoresis , free flow electrophoresis , protein filament , gel electrophoresis , serine , threonine , isoelectric point , biochemistry , polyacrylamide gel electrophoresis , gel electrophoresis of proteins , phosphorylation , enzyme
Wool intermediate filament proteins (IFP) are a subclass of the cytokeratins, a group of structural proteins which form intermediate filaments in many cell types. Post‐translational modifications, such as phosphorylation, play an important role in the control of intermediate filament assembly. Two‐dimensional electrophoresis has previously been used to study the IFP distribution in wools with different physical characteristics. Charge heterogeneity has been observed in Type I and Type II IFP. In a previous study, two‐dimensional electrophoresis of alkaline phosphatase‐treated wool protein extracts was used to show that Type II IFP are phosphorylated. To facilitate post‐separation analysis, micropreparative two‐dimensional electrophoresis was used to separate milligram quantities of wool protein. Direct phosphoamino acid analysis has confirmed the presence of phosphorylation on serine residues on Type II IFP, whose identity was confirmed by amino acid compositional analysis. The isoelectric points of Type I IFP are very similar and they do not separate completely on the commercially available pH 4–7 immobilized pH gradients (IPG) used in this study. In situ tryptic digestion followed by automated Edman sequencing of the high performance liquid chromatography (HPLC)‐separated peptides was used to confirm the identity of this group as Type I IFP. To improve the separation of the Type I IFP it will be necessary to use narrow range IPGs such as Immobiline DryPlates which are available from Pharmacia Biotech, in the pH ranges 4.2–4.9, 4.5–5.4, 5.0–6.0 and 5.6–6.6.