134 Journal of the North Atlantic Special Volume 3 134 Introduction Over the course of the overall Greenland Isotope Project, approximately 400 bone samples were prepared for stable isotope and some for radiocarbon dating measurement. Since reliable isotopic measurements of ancient bone require careful extraction of bone collagen, we describe here in detail the methods employed at the SFU Archaeometry Laboratory for this purpose. At the outset, the method of collagen extraction employed was essentially that of Brown et al. (1988), but during the course of the project, this method was modified to reduce bone sample sizes from ≈100–200 mg to ≈15–25 mg (for stable isotope measurement) and ≈40–80 mg (for radiocarbon measurement) and to reduce processing time from ≈5 to 2 days. Prior to its implementation, we thoroughly tested this new collagen-extraction method with well-documented laboratory standards to ensure that the resulting collagen extract precisely reproduced the known values. We describe here the “new” sampling method and processing procedures, including general qualitative observations of each stage of the extraction process for a typical, well-preserved bone sample. We go into considerable detail, as some of these seemingly trivial observations are quite useful in quality control. Collagen Extraction Protocol In short, our standard collagen-extraction procedure is designed to extract from ancient bone only high molecular-weight, insoluble remnants of the autochthonous collagen polymer. The procedure begins by soaking the bone sample in weak acid to remove bone apatite and any post-depositional mineralization. The insoluble proteinaceous remnants are rendered soluble in very weak acid by shaking at a modest temperature for several hours. The resultant solution is then ultra-filtered and freeze-dried to isolate and concentrate the high molecular-weight protein. A detailed description of these procedures follows. Bone sampling We prefer the bone sample to be in the form of small particles of fairly uniform shape and size. By sampling in this way, we maximize the surface area available for reaction with the acids, which increases the rates of demineralization and dissolution. The bone is sampled by using a low-speed drilling tool (NSK Nakanishi, Inc.) as used in the jewellery and model-making industries. This tool can be fitted with either burs or drills to remove small shavings of the bone. It must be used at low speed to prevent “burning” the collagen through excessive friction at the point of contact. Well-preserved bone gives off a characteristic odor during sampling. If the drill speed is too high, the bone will become brown at the point of contact with the bur and there is a burnt quality to the odor produced. Prior to sampling, any contamination such as adhering soil particles, plant material, soft tissue, etc., is removed from the bone by manually scrubbing the surface and/or briefly sonicating in distilled water. If the latter cleaning method is used, the bone must be dried thoroughly prior to drilling. The specific area of bone to be sampled is then typically milled with a spherical carbide bur (3–5 mm diameter) to remove the surface bone and expose fresh interior material. These surface drillings are discarded, and the underlying bone is sampled with the spherical bur or a 1–2 mm carbide twist drill (standard HSS tools also work well, but they dull quickly in bone). A major advantage here is that the “hole” left in the bone is only a few millimeters in diameter. Not only is this much less destructive, but it also means that the best-preserved part of the bone can be selected for measurement. Drillings obtained in this manner from well-preserved, compact, cortical bone are typically white to light brown in color, and when examined under a low-power dissecting microscope, they have the appearance of fine wood shavings. Burs and drills are scrubbed with a small wire brush and rinsed with water to clean them between samples. It is preferable to examine them microscopically after cleaning, as tiny particles tend to cling tenaciously to the tool. Appendix 1: SFU Archaeometry Laboratory Methods Cheryl M. Takahashi1,* and D. Erle Nelson1 Abstract - The protocol for collagen extract used in the Greenland Isotope Project as discussed in the papers comprising the Journal of North Atlantic Special Volume 3, Greenland Isotope Project: Diet in Norse Greenland AD 1000–AD 1450, is described here in detail. Special Volume 3:134–135 Greenland Isotope Project: Diet in Norse Greenland AD 1000–AD 1450 Journal of the North Atlantic 1Simon Fraser University, Department of Archaeology. Burnaby, Canada BC. *Corresponding author - c_takahashi@sfu.ca. 2012 2012 C.M. Takahashi and D.E. Nelson 135 Demineralizing the bone The bone drillings are weighed into Pyrex culture tubes (13- x 100-mm tubes for samples >50 mg or 16- x 100-mm tubes for samples 50–100 mg) with Teflon-lined screw caps. For well-preserved samples, one can expect a collagen yield in the range of 10–20%, so only 15–25 mg of bone is required to obtain sufficient collagen for stable isotope measurement and about 40–80 mg for AMS measurement. Demineralization is achieved by soaking the drillings in 0.25 N hydrochloric acid (HCl) (≈5 ml for samples <50 mg; ≈8 ml for samples 50–100 mg) in an ultrasonic bath (ULTRAsonik 3Q/H, J.M. Ney Co.) for 20 minutes at maximum power and degas settings. Samples are resuspended at 5-minute intervals to ensure maximum surface-area exposure to the acid. After sonication, the samples are centrifuged and the supernatent is discarded. To rinse away residual dissolved minerals, the remaining insoluble material is resuspended in 0.01 N HCl, centrifuged again, and the acid decanted. Collagen dissolution After demineralization, the insoluble collagen is dissolved by rocking the sample with 0.01 N HCl (4 ml for samples <50 mg; 8 ml for samples 50–100 mg) in a thermal rocker (Lab-line Instuments, Inc.) set to 58 °C and a moderate speed setting. Dissolution time varies with the degree of collagen preservation, so it is important to monitor the samples during this stage of the extraction process. Heating the samples too long will degrade the collagen to low molecular weight fragments and reduce the yield of the high molecular weight fraction. Samples should be removed from the thermal rocker when no more insoluble material is visible and the solution is clear. In general, dissolution takes ≈16 to 24 hours. Concentration of the high molecular weight fraction When dissolution is complete, each sample is suction-filtered through a glass fiber filter (GF/A, Whatman, Inc.) to remove any large particulates. The filtrate is collected in a 4-ml or 15-ml ultrafiltration device with a nominal 30-kiloDalton molecular-weight limit membrane (Ultrafree-4, Ultrafree-15, Millipore Corporation). As this (and other) ultrafilter membranes contain a small amount of glycerin as a humectant, it must be removed prior to use. This cleansing is done by passing three volumes of ultra-pure water through the membrane and discarding the filtrates. (Note: After this project was completed, the manufacturer changed the formulation of their Ultrafree-4 filtration devices to one that we have found unsuitable. We now use a different product [Vivaspin 4-ml Concentrator, Vivascience, Inc.] with the same description and characteristics as the original Ultrafree-4. Care must be taken in selecting these devices, as they are not all equal.) The ultrafilter is centrifuged at 3200 r.c.f. to concentrate the >30-kD size fraction to <500 μl. The filtrate (<30-kD fraction) is transferred to a glass vial and set aside. The >30-kD fraction is desalted by reconstituting to 2 x 4 ml (15 ml for Ultra-free-15) with ultra-pure water and re-concentrating to <500 ul. This process is repeated once more to ensure the removal of all low molecular-weight solutes. The filtrates from each desalting step are pooled with that from the initial filtration. The final >30-kD concentrate is transferred to a tared glass vial (we find a “1-dram” vial convenient). Samples with high collagen yield will be slightly viscous and have a tendency to foam when pipetted. Samples are frozen in liquid nitrogen and lyophilized. Lyophilized collagen extracted from well-preserved bone is white to light brown in color and has a fine “velveteen” texture and a sponge-like appearance. If the >30-kD fraction yields no collagen, we concentrate the <30-kD fraction (which had been set aside earlier) with a 10-kD molecular-weight limit ultrafilter. If the lyophilized >10-kD fraction has the appearance of well-preserved collagen as described above, it may be submitted for measurement. Literature Cited Brown, T.A., D.E. Nelson, J.S. Vogel, and J.R. Southon. 1988. Improved collagen extraction by modified Longin method. Radiocarbon 30:171–177.