Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II.
Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.
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Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Pervaiz
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Lee
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D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats / Q000276:immunology; D007106:Immune Sera / Q000276:immunology*; D007114:Immunization; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000276:immunology*; D011817:Rabbits; D012709:Serum Albumin / Q000276:immunology
D000941:Antigens; D007106:Immune Sera; D012709:Serum Albumin; D010749:Phosphoprotein Phosphatases