Z. Deyl et al., QUANTITATION OF COLLAGEN TYPE-I, TYPE-III AND TYPE-V IN TISSUE-SLICESBY CAPILLARY ELECTROPHORESIS AFTER CYANOGEN-BROMIDE SOLUBILIZATION, Journal of chromatography B. Biomedical sciences and applications, 689(1), 1997, pp. 181-194
Citations number
28
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
A method for the determination of the proportions of major fiber-formi
ng collagens (types I, III and V) in soft connective tissue was elabor
ated. The method is based on the release of insoluble collagen by CNBr
with subsequent separation of the arising peptides. For routine appli
cation the peptides are separated by capillary electrophoresis (50 mM
phosphate pH 2.5, 15 kV, 50 degrees C, 70/60 cmX70 mu m I.D. capillary
with UV detection at 200 nm). Quantitation of collagen type I can be
done either on the basis of spiking the sample with a peptide mixture
obtained from a known amount of collagen type I, or by spiking the sam
ple with an equimolar mixture of the two peptides [alpha(1)(I)CB2 and
alpha(1)(I)CB4] (constituting a fused peak) along with alpha(1)(III)CB
2 and alpha(1)(V)CB1. Compared to the previously published methods the
procedure is faster and does not require isolation of marker peptides
by tedious chromatographic procedures in a preceding preparatory step
. Good results are obtained within a wide range of run buffer concentr
ations and applied voltages; conversely, intensive cleaning of the cap
illary after every three runs is recommended with a new capillary afte
r 20-30 runs.