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 p...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 1998-06, Vol.247 (2), p.312-314
Main Authors: Praul, Craig A., Brubaker, Kristen D., Leach, Roland M., Gay, Carol V.
Format: Article
Language:English
Subjects:
Animals
Biotin - analysis
Biotin - metabolism
Blotting, Western - methods
Carboxy-Lyases - chemistry
Carboxy-Lyases - isolation & purification
Carboxy-Lyases - metabolism
Chickens
chondrocyte
Chondrocytes - metabolism
endogenous biotin
In Vitro Techniques
Male
Molecular Weight
Oligopeptides - chemistry
Oligopeptides - metabolism
osteoblast
Osteoblasts - metabolism
osteoclast
Osteoclasts - metabolism
Proteins - chemistry
Proteins - isolation & purification
Proteins - metabolism
Pyruvate Carboxylase - chemistry
Pyruvate Carboxylase - isolation & purification
Pyruvate Carboxylase - metabolism
Streptavidin - metabolism
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Summary: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.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1998.8757