Immunoblotting studies on artifactual contamination of enamel homogenates by albumin and other proteins.
The reason for the presence of albumin and other serum, cytoskeletal, cytosolic, and extracellular matrix proteins in enamel fractions was investigated by immunoblotting using homogenates prepared from freeze-dried and freshly dissected rat incisors, and antibodies capable of resolving at least 1 ng of the primary antigen. The data indicated that most of the 16 antibodies examined in this study reacted with antigens present only within "cell" homogenates (enamel organ cells + adhering labial connective tissue and blood vessels). One exception was rat serum albumin which was detected routinely in enamel homogenates prepared from freshly dissected, wiped incisors but rarely within enamel homogenates prepared from freeze-dried incisors. Another exception was calbindin-D 28 kDa which was consistently found within secretory stage enamel homogenates irrespective of preparative technique. A third exception was enamel proteins (amelogenins) which were enriched in secretory and early maturation stage enamel homogenates compared with cell homogenates and distributed as multiple molecular weight, antigenic bands in enamel homogenates (14-30 kDa), but mostly as a single antigenic band in cell homogenates (near 27 kDa). Overall, the results of this study suggest that developing rat incisor enamel naturally contains few exogenous proteins such as albumin. High concentrations of albumin (or other serum proteins) in crude homogenates, or purified fractions, derive mostly from blood and/or tissue fluids soaking into the enamel during sample preparation. This type of artifact can be avoided by using freeze-dried teeth for biochemical analyses.
Chen
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Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
Nanci
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Smith
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D053523:Amelogenin; D000818:Animals; D000906:Antibodies; D016477:Artifacts; D001798:Blood Proteins / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D003598:Cytoskeletal Proteins / Q000032:analysis; D003743:Dental Enamel / Q000737:chemistry*; D003746:Dental Enamel Proteins / Q000032:analysis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005612:Freeze Drying; D006801:Humans; D007180:Incisor / Q000648:ultrastructure; D008297:Male; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000032:analysis
D053523:Amelogenin; C507201:Amelx protein, rat; D000906:Antibodies; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D003746:Dental Enamel Proteins; D016326:Extracellular Matrix Proteins; D011506:Proteins; D012709:Serum Albumin