The use of preparative polyacrylamide gel electrophoresis and electroelution for purification of mucus glycoproteins.
This paper describes a novel technique for purifying glycoproteins from porcine gastric mucus by preparative polyacrylamide gel electrophoresis and electroelution. The method is based on the observation that the high-molecular-weight buffer/SDS-soluble mucins do not penetrate through the polyacrylamide gel, but remain on the gel surface. Mucus solution extracted with 6 M urea was fractionated on Sepharose CL-2B column and Vo peak mucin was submitted to purification by preparative polyacrylamide gel electrophoresis (22 h). Nonpenetrated mucin layer was electroeluted from the gel after the reversing of electrode polarity (3 h). A comparison of mucin preparations purified by our method and by CsCl density gradient centrifugation indicated that the GalNAc/protein and GalNAc/DNA ratios were three times higher than those of the first method. The method is a relatively short and efficient procedure and yields pure mucin preparation free of contaminating proteins and nucleic acids.
Paszkiewicz-Gadek
A
A
Department of General and Organic Chemistry, Medical Academy 15-222 Bialystok, Mickiewicza, Poland.
Gindzieński
A
A
Porowska
H
H
D000116:Acetylgalactosamine / Q000032:analysis; D000117:Acetylglucosamine / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000032:analysis; D005690:Galactose / Q000032:analysis; D005752:Gastric Mucins / Q000737:chemistry / Q000302:isolation & purification*; D019791:Guanidine; D006146:Guanidines; D008358:Mannose / Q000032:analysis; D009093:Mucus / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D014508:Urea
D005752:Gastric Mucins; D006146:Guanidines; D005643:Fucose; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D019791:Guanidine; D000116:Acetylgalactosamine; D008358:Mannose; D000117:Acetylglucosamine; D005690:Galactose