Ab. Chen et al., COMPARISON OF AMPHOLYTES USED FOR SLAB GEL AND CAPILLARY ISOELECTRIC-FOCUSING OF RECOMBINANT TISSUE-TYPE PLASMINOGEN-ACTIVATOR GLYCOFORMS, Journal of chromatography, 744(1-2), 1996, pp. 279-284
Citations number
8
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
Four commercial ampholytes: Ampholine and Pharmalyte (Pharmacia Biotec
h), Bio-Lyte (Bio-Rad) and Servalyt (Serva) were evaluated for their a
bility to resolve recombinant tissue-type plasminogen activator (rt-PA
) glycoforms by isoelectric focusing (IEF) and capillary IEF (cIEF). E
ach brand of ampholytes focused rt-PA into 3-4 major and 5-6 minor ban
ds on slab gel electrophoresis. Visually, focused bands stained with C
oomassie Blue appeared to be similarly resolved by all the ampholytes
except for Ampholines, where the bands were closely grouped and more i
ntensely stained. When cIEF was performed, Pharmalytes and Ampholines
resolved rt-PA glycoforms consistent with the slab gels. No discernibl
e peaks were detected during cIEF of rt-PA using Servalyts or Bio-Lyte
s. UV spectrophotometric scans of the components used for cIEF showed
that Servalyts absorbed intensely over a range which overlapped the de
tector bandpass. Bio-Lytes showed absorption over a narrower UV range
but still overlapped the detector bandpass, thus preventing the discer
nment of protein peaks. For this cIEF system the best ampholytes were
Ampholines and Pharmalytes.