Detection of arylsulfatase A activity after electrophoresis in polyacrylamide gels: problems and solutions. |
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Authors: | P L Chang S R Ballantyne R G Davidson |
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Institution: | Department of Pediatrics, Room 3N17, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario L8S 4J9, Canada |
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Abstract: | When arylsulfatase extracted from normal human skin fibroblasts was electrophoresed in polyacrylamide gels with a Tris-glycine buffer at pH 8.4–8.6, two problems occurred. First, no arylsulfatase A activity was detected. Second, an artifactual fluorescent spot was generated when the gels were stained for arylsulfatase activity with 4-methylumbelliferyl sulfate as substrate. The artifact simulated arylsulfatase A activity in mobility but also appeared when 4-methylumbelliferyl substrates for other enzymes were used. It can be eliminated by prerunning or prolonged storage of the gets before use. The arylsulfatase A activity, however, could be recovered only when a low pH buffer system (pH 58–68) was used for electrophoresis. The highest percentage recovery (70%) of activity was obtained in acrylamide gels polymerized with ammonium persulfate, prerun for 0.5 h before use and electrophoresed with an anode buffer of acetic acid-triethanolamine at pH 5.8. |
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