Detection of endogenous biotin-containing proteins in bone and cartilage cells with streptavidin systems.
When utilizing streptavidin systems with Western blots of chondrocyte, osteoblast and osteoclast lysates, proteins of the molecular weights 116 kDa, 75 kDa and 67 kDa were observed to be bound by streptavidin alone. Streptavidin binding could not be blocked by pre-incubation with an RGD containing peptide. The same proteins were bound by ExtrAvidin which lacks the RGD sequence present in streptavidin. Pre-incubation with free biotin completely abolished the binding of both streptavidin and ExtrAvidin. The three proteins observed are believed to be the biotin containing carboxylases: pyruvate carboxylase, 3-methylcrotonyl carboxylase, and propionyl carboxylase. The findings of this study underscore the need to apply vigorous controls to distinguish between endogenous biotinylated proteins and biotin used as a means to amplify avidin detection systems since a wide variety of proteins with relevance to bone and cartilage biology have molecular weights similar to the biotin carboxylases.
Praul
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Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Brubaker
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Leach
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Gay
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D000818:Animals; D001710:Biotin / Q000032:analysis / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002262:Carboxy-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002645:Chickens; D019902:Chondrocytes / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D009842:Oligopeptides; D011506:Proteins; D001710:Biotin; C047981:arginyl-glycyl-aspartic acid; D019809:Streptavidin; D002262:Carboxy-Lyases; D011766:Pyruvate Carboxylase