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.