New detection method after cellulose acetate membrane isoelectric focusing: activity staining for lactate dehydrogenase, detection for sugar chains using lectin and simple western blotting.
To extend the clinical applications for cellulose acetate (CA) membrane isoelectric focusing, we designed two detection methods and two blotting methods that improve the characteristics of the CA membrane; that is, detection of the lactate dehydrogenase (LD) isozyme on the CA membrane, detection of the sugar chain in glycoprotein on the CA membrane, and simple and rapid methods for western blotting. In the detection of the LD isozyme, when 50% saturated ammonium sulfate solution was used as a fixing solution, five main LD bands and seven sub LD bands were detected clearly. By treating the CA membrane with 5% sulfosalicylic acid, glycoprotein was fixed on the CA prior to detection of the sugar chain of glycoprotein using Lens Culinaris (LCA), Phasseolus Vulgaris-L(4 )(PHA-L(4)), Maackia Amurensis (MAM), and Sambucus Siedoldiana (SSA) lectins. Using this procedure, clear electrophoretic patterns were obtained. As simple and rapid methods for blotting proteins on polyvinylidene difluoride membranes, we designed natural contact blotting and suction blotting techniques. The natural contact blotting method did not need special apparatus. The suction blotting method completed protein blotting for 10 minutes. By both methods, clear electrophoretic patterns were obtained.
Iijima
S
S
Division of Toxicology, Kyoritsu College of Pharmacy, Tokyo, Japan. iijima-sr@kyoritsu-ph.ac.jp
Kimura
M
M
Shiba
K
K
D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D002482:Cellulose / Q000031:analogs & derivatives*; D002624:Chemistry, Clinical / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000737:chemistry; D037102:Lectins; D008567:Membranes, Artificial; D008970:Molecular Weight; D010835:Phytohemagglutinins; D013396:Suction
D002241:Carbohydrates; D006023:Glycoproteins; D007527:Isoenzymes; D037102:Lectins; D008567:Membranes, Artificial; D010835:Phytohemagglutinins; C100156:leukoagglutinins, plants; C005062:acetylcellulose; D002482:Cellulose; D007770:L-Lactate Dehydrogenase