Status of Cys Residues in the Covalent Structure of Botulinum Neurotoxin Types A, B, and E

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10, and 8 Cys residues, respectively, as deduced from nucleotide sequences [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345–2354]. Each of the 150-kDa NTs has at least one disulfide; but type B, like types A and E, may have two disulfides. Using two different chemical reagents, we studied the status of the Cys residues in these three proteins after (i) the final anion exchange chromatographic step in their purification (fresh NT), (ii) 24 hr storage at 8°C, (iii) precipitation with ammonium sulfate (precipitated NT), and (iv) dissolving the precipitated NT in 6 M guanidine·HCl. In all three NT serotypes the number of Cys residues titrated with 5,5-dithiobis-2-nitrobenzoic acid (DTNB) as free -SH groups varied, depending upon the absence or presence of EDTA added to the chromatography buffer, storage condition, age, and presence of the denaturant. Titration of 9.5–10 and 5.4–6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and partial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformational change(s). When the precipitated NTs were dissolved in 6 M guanidine·HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4–8.3 Cys in type A NT were titratable with DTNB. Iodoacetamide modification of precipitated NT types A, B, and E carboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine·HCl in the absence of any added reducing agent. We propose that -S-S- cleavage mediated by the -SH/-S-S- exchange observed in vitro after unfolding the NTs (also unfolded by 2 M guanidine·HCl or urea) possibly mimicks a similar exchange process inside the endosomes, where the NTs are thought to undergo conformational changes, resulting in the reductive cleavage of the interchain disulfide between the 50-kDa light and 100-kDa heavy chain, which in turn releases the light chain and allows its egress out of the endosomes into the cytosol.     

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

The non-mineral component of bone matrix consists of 90% collagenous, 10% non-collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass-spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty-one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states.     

Protein synthesis during cold shock in barley tissues

Summary In barley (Hordeum vulgare L.) seedlings, a temperature step-down from 24 °C to 6°C (cold shock) determined a reduction in the incorporation of labeled aminoacids and modified the electrophoretic pattern of total proteins. At 6 °C some new proteins appeared and others were intensified (cold shock-induced proteins= CSPs); meantime, few proteins disappeared or were curtailed (cold-repressed proteins=CRPs). The majority of the proteins of the seedlings were labeled at about the same rate both at 6 °C and 24 °C, whereas at 0 °C only the cold shock proteins and a few others were detectable. The cold shock-induced variations of the protein profile differed in roots and in seed remnants which showed only some of the CSPs detected in roots. Total protein synthesis of barley genotypes Onice and Georgie, which have respectively a winter and spring growth habit, were similarly inhibited by a temperature drop. The two genotypes, however, showed some differences in the CSPs and CRPs pattern. Because Onice and Georgie have also a different thermotolerance, the hypothesis can be made that in barley specific CSPs are involved in conferring various degrees of cold resistance.     

Microspectroscopic evidence of cretaceous bone proteins

Low concentrations of the structural protein collagen have recently been reported in dinosaur fossils based primarily on mass spectrometric analyses of whole bone extracts. However, direct spectroscopic characterization of isolated fibrous bone tissues, a crucial test of hypotheses of biomolecular preservation over deep time, has not been performed. Here, we demonstrate that endogenous proteinaceous molecules are retained in a humerus from a Late Cretaceous mosasaur (an extinct giant marine lizard). In situ immunofluorescence of demineralized bone extracts shows reactivity to antibodies raised against type I collagen, and amino acid analyses of soluble proteins extracted from the bone exhibit a composition indicative of structural proteins or their breakdown products. These data are corroborated by synchrotron radiation-based infrared microspectroscopic studies demonstrating that amino acid containing matter is located in bone matrix fibrils that express imprints of the characteristic 67 nm D-periodicity typical of collagen. Moreover, the fibrils differ significantly in spectral signature from those of potential modern bacterial contaminants, such as biofilms and collagen-like proteins. Thus, the preservation of primary soft tissues and biomolecules is not limited to large-sized bones buried in fluvial sandstone environments, but also occurs in relatively small-sized skeletal elements deposited in marine sediments.     

Extracellular matrix proteins involved in bone induction are vitamin D dependent

Subcutaneous implantation of demineralized diaphyseal bone matrix into allogeneic rats results in local formation of cartilage and bone. However, implantation of demineralized bone matrix obtained from rachitic rats did not induce bone. Rachitic bone matrix was therefore dissociatively extracted with 4 M guanidine HCl and then reconstituted with an inactive collagenous residue of control as carrier. Such reconstituted materials also lacked bone inductive potential. On the other hand, reconstitution of guanidine HCl extracts of control bone matrix with inactive vitamin D deficient matrix did result in bone induction. Partial purification (fractions containing proteins (less than 50,000 daltons) of the guanidine HCl extract from rachitic rats on Sepharose CL-6B followed by reconstitution with inactive collagenous residues resulted in a weak (25% of control) inductive response. These observations imply that bone inductive proteins are vitamin D dependent and are reduced in matrix obtained from rachitic rats.     

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve <Emphasis Type="Italic">Pecten maximus</Emphasis> in Human Articular Chondrocytes— Application for Cartilage Engineering

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.     

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.Download video file.^{(49M, mov)}     

Importance of geometry of the extracellular matrix in endochondral bone differentiation

Subcutaneous implantation of coarse powders (74-420 micron) of demineralized diaphyseal bone matrix resulted in the local differentiation of endochondral bone. However, implantation of matrix with particle size of 44-74 micron (Fine matrix) did not induce bone. We have recently reported that the dissociative extraction of coarse matrix with 4 M guanidine HCl resulted in a complete loss of the ability of matrix to induce endochondral bone; the total loss of biological activity could be restored by reconstitution of extracted soluble components with inactive residue. To determine the possible biochemical potential of fine matrix to induce bone, the matrix was extracted in 4 M guanidine HCl and the extract was reconstituted with biologically inactive 4 M guanidine HCl-treated coarse bone matrix residue. There was a complete restoration of the biological activity by the extract of fine matrix upon reconstitution with extracted coarse matrix. Polyacrylamide gel electrophoresis of the extract of fine matrix revealed similar protein profiles as seen for the extract of coarse matrix. Gel filtration of the 4 M guanidine HCl extract of fine powder on Sepharose CL-6B and the subsequent reconstitution of various column fractions with inactive coarse residue showed that fractions with proteins of 20,000-50,000 mol wt induced new bone formation. These observations demonstrate that although fine bone matrix contains, osteoinductive proteins, matrix geometry (size) is a critical factor in triggering the biochemical cascade of endochondral bone differentiation. Mixing of coarse matrix with Fine results in partial response and it was confined to areas in contact with coarse particles. The results imply a role for geometry of extracellular bone matrix in anchorage-dependent proliferation and differentiation of cells.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Sorophores of<Emphasis Type="Italic">Lygodium</Emphasis> Sw. (Schizaeaceae) from the Late Cretaceous of New Jersey

We report here on a series of specimens of charcoalified sorophores with characteristics of the extant fern genusLygodium (Schizaeaceae) collected from sediments of the Raritan Formation (Late Cretaceous). Each elongate lobed fertile pinnule (sorophore) is flattened and bears alternately arranged sporangia on one surface. Each sporangium is covered by an indusium continuous with the margin of the lamina. Sporangia are oblong in shape, short stalked, and have an apical annulus formed by a single ring of radiating cells that dehisces longitudinally. The sporangial cap or distal face is formed by only one cell. All of these features are characteristic of the extant genusLygodium. Small numbers of trilete, psilate spores are found in the sporangia. Megafossils assignable toLygodium are known from the Upper Cretaceous of North America and Germany with worldwide distribution during the Tertiary. The newLygodium fossils are compared with others previously referred to the genus.     

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.Download video file.^{(112M, mp4)}     

Molecular and cytological characteristics of nuclear DNA and chromatin for angiosperm systematics: Basic data forHelianthus annuus (Asteraceae)

As a contribution for the study of systematic and evolutionary relationships it is suggested to analyze nuclear DNA and chromatin by means of CsCl ultracentrifugation, thermal denaturation and renaturation, scanning densitometry, and (ultra)structural analyses. Relevant data have been obtained forHelianthus annuus as a first example.The 2C DNA content of four cultivars ofHelianthus annuus L. was calibrated by comparative measurement withAllium cepa nuclei using a scanning densitometer in on-line operation with a computer. Significant infraspecific variation could be detected: cvar. Amerikanische Riesen displayed 6.1 pg, cvar. Gefüllte Vielblütige 9.9 pg, cvar. Russian Mammoth 8.9 pg, and a Heidelberg strain 8.7 pg.The buoyant density in neutral CsCl was determined for cvar. Amerikanische Riesen to be 1.695 g · cm^–3; this corresponds to an average GC content of 35.1%. Thermal denaturation revealed a melting temperature of 86.4°C. Derivative thermal denaturation profiles led to the detection of several distinct DNA fractions.The species-specific nuclear structure is of the chromonematic type, but in differentiated cells the chromatin fibers may be more decondensed so that a chromomere-interchromomere structure appears. The heterochromatin constitutes an average of 4.5% of the total genome. Chromatin ultrastructure is characterized by a diffuse distribution of chromatin threads and patches. Nucleosomes of 110 Å diameter can be recognized.The data are discussed (a) in relation to findings on DNA variation in other plants, (b) in relation to the systematic usefulness and further characterization of nuclear DNA and chromatin, and (c) in relation to tissue-specific and functional variation of the species-specific chromatin structure.     

Production of biologically active hirudin in plant seeds using oleosin partitioning

A plant oleosin was used as a carrier for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin carrier. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Conversion of phenoloxidase and peroxidase indicators in individual haemocytes of Mytilus edulis specimens and isolation of phenoloxidase from haemocyte extract

Haemocytes oxidized 3-amino-9-ethylcarbazole and other peroxidase indicators such as 3,3-diaminobenzidine·4HCl, 3,3,5,5 tetramethylbenzidine·2HCl and 4-chloro-1-naphthol without addition of H₂O₂ indicating that the reaction was possibly not caused by a peroxidase. As these chromogens were also converted by a mushroom phenoloxidase in the absence of H₂O₂, cell smears were incubated with known substrates of phenoloxidases. One of these, l-dopa, caused strong melanin formation in several haemocytes and the reaction could be blocked by a variety of inhibitors including KCN, NaF, 1-phenyl-2-thiourea, cysteine, glutathione, ascorbic acid and HgCl₂. The enzymatic activity was isolated using a concanavalin A column and separated into two fractions with an ion-exchange cartridge. The molecular weights of the glycoproteins were estimated to be 381±13.7 kDa and 316±11.1 kDa. After isoelectric focusing of a haemocyte extract and the two ion-exchange peaks, seven enzyme bands were detected with isoelectric points between pH 5.0 and 5.5. The isolated enzyme fractions both converted 3,3,5,5-tetramethylbenzidine·2HCl best at pH 5–6 and l-dopa at pH 7.0 without addition of H₂O₂. Heat-treated cells lost their enzymatic activity; however, a group of haemocytes still bound preoxidized 3-amino-9-ethylcarbazole (= AECox). Also, some of the phenoloxidase inhibitors mentioned above blocked this non-enzymatic staining reaction. About 30–57% of haemocytes from individual mussels were AECox-positive, whereas Mytilus specimens without phenoloxidase-containing cells often occurred. Haemocytes containing this enzyme exhibited a high mobility and a large percentage of them belonged to a cytotoxic cell population.Abbreviations AEC 3-amino-9-ethylcarbazole - AECox preoxidized AEC - BSA bovine serum albumin - 4CN 4-chloro-1-naphthol - DAB 3,3-diaminobenzidine·4HCl - DMFA dimethylformamide - EDTA ethylenediaminetetra-acetic acid - LGT Iow gelling temperature - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonylfluoride - PTU 1-phenyl-2-thiourea - RBC red blood cells - TCA trichloroacetic acid - TMB 3,3,5,5-tetramethylbenzidine·2 HCl     

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Download video file.^{(49M, mov)}     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

Synthesis of serum proteins by cultured aggregates from endodermal cells of the area opaca of the primitive streak chick embryo

Summary The pattern of serum protein synthesis and secretion in aggregates of extraembryonic endoderm cells (EEC) from the area opaca of primitive streak chick embryos was studied. EEC aggregates were cultured for various time intervals and serum proteins were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Serum proteins were identified based on their comigration with reference proteins from 4 day chick embryo serum and with reference proteins from egg white albumen and chicken serum. A number of serum proteins were detected in EEC aggregates including: two variants of immunoglobulin (IgG), four variants of transferrin, a protein with a molecular weight of 66 500 which may correspond to globulins, prealbumin, and a protein with a molecular weight of 38 600 (serum protein 11) which remains unidentified. These proteins were also detected in the culture medium. The banding profiles of EEC extracts and culture medium were compared over various time intervals of culture (6, 18 and 30 h). The IgGs, transforms and serum protein 11 decreased in concentration in EEC extracts over the culture interval. These proteins as well as prealbumin, were detected in the culture medium. A number of proteins were synthesized by EEC, as determined by radiolabelled amino acid incorporation. All of the labelled serum proteins were detected in the culture medium, not in EEC extracts. These results suggest that serum proteins are synthesized by EEC then rapidly released into the medium. Labelled serum proteins detected in the culture medium include prealbumin and an unidentified serum protein (serum protein 14) which migrates with the tracking dye, both synthesized early in culture (6 h), and transferrin which was synthesized later (18 h) during culture.     

Collagen and non-collagenous proteins in different mineralization stages of human femur

Mineralized tissues exhibit varying degrees of mineralization in different areas within the same bone. Using the technique of density gradient fractionation, bone powder from the diaphysis of human femur has been separated in different fractions corresponding to the degree of mineralization. Isolated bone fractions were analysed for their content in collagen and non-collagenous proteins. The results showed marked differences between compact and spongy bones, this latter containing higher proportions of little mineralized bone particles than the former (p less than 0.01). The ethylenediaminetetra-acetic acid (EDTA) extractability and the bone matrix size decreased relative to the decrease in specific gravity of bone particles. Among the matrix components of different fractions, sialoprotein consistently increased with the increase in specific gravity while proteoglycan decreased in reverse manner to the increase in collagen. However, in the most mineralized fraction (specific gravity: 2.33 g/cm3), the proteoglycan amount increased while collagen decreased. In conclusion, this study of bone maturation in human femur confirms the suitability of the technique of density gradient fractionation in the studies of bone matrix-mineral interactions. Apart from the fraction with the highest specific gravity, the analytical results obtained in fractions are similar to those observed in age-related bone changes, suggesting that the increase in mineralization degree of bone particles may be related to their age.     

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.