Br. Herbert et al., CHARACTERIZATION OF WOOL INTERMEDIATE FILAMENT PROTEINS SEPARATED BY MICROPREPARATIVE 2-DIMENSIONAL ELECTROPHORESIS, Electrophoresis, 18(3-4), 1997, pp. 568-572
Wool intermediate filament proteins (IFP) are a subclass of the cytoke
ratins, a group of structural proteins which form intermediate filamen
ts in many cell types, Post-translational modifications, such as phosp
horylation, play an important role in the control of intermediate fila
ment assembly. Two-dimensional electrophoresis has previously been use
d to study the IFP distribution in wools with different physical chara
cteristics. Charge heterogeneity has been observed in Type I and Type
II IFP. In a previous study, two-dimensional electrophoresis of alkali
ne phosphatase-treated wool protein extracts was used to show that Typ
e II IFP are phosphorylated. To facilitate post-separation analysis, m
icropreparative two-dimensional electrophoresis was used to separate m
illigram quantities of wool protein. Direct phosphoamino acid analysis
has confirmed the presence of phosphorylation on serine residues on T
ype II IFP, whose identity was confirmed by amino acid compositional a
nalysis, The isoelectric points of Type I IFP are very similar and the
y do not separate completely on the commercially available pH 4-7 immo
bilized pH gradients (IPG) used in this study, In situ tryptic digesti
on followed by automated Edman sequencing of the. high performance liq
uid chromatography (HPLC)-separated peptides was used to confirm the i
dentity 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 Immob
iline 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.