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Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte‐immobilized pH gradient polyacrylamide gels
Author(s) -
Khoo Kong Soo,
Beeley Josie A.
Publication year - 1990
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.1150110610
Subject(s) - immobilized ph gradient , chemistry , chromatography , isoelectric focusing , isoelectric point , polyacrylamide , coomassie brilliant blue , centrifugation , urea , staining , biochemistry , polymer chemistry , medicine , pathology , enzyme
Isoelectric focusing of human salivary proteins with carrier ampholyte‐isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time‐consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (> 50 μg) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4–9 and pH 3.5–5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4–9 gels it gave improved resolution of protein bands, whereas in pH 3.5–5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G‐250 or a silver procedure. Using the latter, 25–30 well‐resolved bands were formed on a pH 4–9 gel loaded with 20 μg of proteins. The method offers considerable advantages compared with carrier ampholyte‐isoelectric focusing.