12 research outputs found
Empirical Evaluation of Bone Extraction Protocols
The application of high-resolution analytical techniques to characterize ancient bone proteins requires clean, efficient extraction to obtain high quality data. Here, we evaluated many different protocols from the literature on ostrich cortical bone and moa cortical bone to evaluate their yield and relative purity using the identification of antibody-antigen complexes on enzyme-linked immunosorbent assay and gel electrophoresis. Moa bone provided an ancient comparison for the effectiveness of bone extraction protocols tested on ostrich bone. For the immunological part of this study, we focused on collagen I, osteocalcin, and hemoglobin because collagen and osteocalcin are the most abundant proteins in the mineralized extracellular matrix and hemoglobin is common in the vasculature. Most of these procedures demineralize the bone first, and then the remaining organics are chemically extracted. We found that the use of hydrochloric acid, rather than ethylenediaminetetraacetic acid, for demineralization resulted in the cleanest extractions because the acid was easily removed. In contrast, the use of ethylenediaminetetraacetic acid resulted in smearing upon electrophoretic separation, possibly indicating these samples were not as pure. The denaturing agents sodium dodecyl sulfate, urea, and guanidine HCl have been used extensively for the solubilization of proteins in non-biomineralized tissue, but only the latter has been used on bone. We show that all three denaturing agents are effective for extracting bone proteins. One additional method tested uses ammonium bicarbonate as a solubilizing buffer that is more appropriate for post-extraction analyses (e.g., proteomics) by removing the need for desalting. We found that both guanidine HCl and ammonium bicarbonate were effective for extracting many bone proteins, resulting in similar electrophoretic patterns. With the increasing use of proteomics, a new generation of scientists are now interested in the study of proteins from not only extant bone but also from ancient bone
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REACTION OF SINGLE-STRANDED-DNA WITH HYDROXYL RADICAL GENERATED BY IRON(II) ETHYLENEDIAMINETETRAACETIC ACID
The survival of organic matter in bone: A review
If bone is considered as a composite of collagen (protein) and bioapatite (mineral), then three pathways of diagenesis are identified: (1) chemical deterioration of the organic phase; (2) chemical deterioration of the mineral phase; and (3) (micro) biological attack of the composite. The first of these three pathways is relatively unusual and will only occur in environments that are geochemically stable for bone mineral. However, because rates of biomolecular deterioration in the burial environment are slow, such bones would yield useful biomolecular information. In most environments, bones are not in thermodynamic equilibrium with the soil solution, and undergo chemical deterioration (path 2). Dissolution of the mineral exposes collagen to biodeterioration, and in most cases the initial phase of dissolution will be followed by microbial attack (path 3). Biological attack (3) also proceeds by initial demineralization; therefore paths 2 and 3 are functionally equivalent. However,in a bone that follows path 3 the damage is more localized than in path 2, and regions equivalent to path 1 may therefore exist outside these zones of destruction. Other biomolecules, such as blood proteins, cellular lipids and DNA, exist within the physiological spaces within bone.For these biomolecules, death history may be particularly important for their survival