Isolation and preparation of collagen for isotopic analysis
Caveat:The procedure below represents a basic outline for preparing bone samples for collagen isotopic analysis. Modification of this procedure may be required for samples of different histories.
Collect samples1. Clean the surface of bone or tooth
2. Collect sample either as a chunk or as a powder (by drilling). If drilling, do not heat the sample too much. Avoid that that sickening dentist office aroma (burning collagen).
3. Place powders in vials. If collecting chunks, place directly into centrifuge tubes.
Decalcify samples1. Weigh out sample to be decalcified, approximately 50 mg. Bone and dentin are ~30% collagen, and we need only 0.7 mg for an isotopic analysis. However since we may be analyzing N and C isotopes on different splits of the collagen (or we may lose the sample) try to get a collagen yield of 6 mg, which means treating ~ 20 mg of raw bone. (If you have smaller amounts of samples scale down protocol accordingly).
2. Place sample in 15 ml glass centrifuge tubes if they will be defatted. If not, they can go into plastic centrifuge tubes.
3. Make a 0.5N HCl solution, using HPLC grade concentrated HCl and distilled/deionized water.
4. Add ~10 ml 0.5N HCl solution to samples.
5. Place caps of Al foil on tubes. If using plastic centrifuge tubes, do not seal tight or the lids might blow.
6. Place samples in 4°C refrigerator or under the fume hood.
7. Allow to decalcify. There are no rules about this. It should take no more than 2 days for powders, and will be faster at room temperature than in the refrigerator. Powders will become pale yellow or beige. Chunks may take longer. If the chunks seem stiff (try to cut them with a scalpel), add new acid and give them another day or two.
8. For powders, centrifuge and remove acid by aspiration. For chunks, just pour off acid.
9. Rinse samples 5x with distilled/deionized water. Again, for powders, centrifuge each time before removing water by aspiration.
Remove humic acids1. Make a 0.1N NaOH solution using NaOH powder/tablets and distilled/deionized water.
2. Add 0.1 M NaOH to samples. Use same amount as for HCl. Allow to sit for 12 to 24 hrs, either at room temperature or in refrigerator.
3. If samples were dark and have turned pale, you are done. If the samples and solutions are brown, centrifuge, aspirate or decant supernant, and refresh NaOH solution.
4. For powders, centrifuge and remove NaOH by aspiration. For chunks, just pour off acid.
5. Rinse samples 5x with distilled/deionized water. Again, for powders, centrifuge each time before removing water by aspiration.
For dentin, enamel or fossil bones, which are not defatted10. Freeze dry samples
11. Remove from freeze drier, store in dessicator.
Defatting (typically only for modern bones)Bones can contain a lot of lipid, which has a different carbon isotope values than collagen. These lipids should be removed prior to analysis. 1. Sample powders or chunks must be glass tubes. Do not use plastic!! Chloroform/methanol will dissolve plastic.
Do all work with chloroform under the fume hood!
2. Make a mixture that is 2.0/1/0.8 v:v methanol/chloroform/water.
3. Add approximately 10 ml methanol/chloroform/water to samples. Figure out some way to agitate samples (shaker/ultrasonic zapping).
4. Decant solution into waste container. Do not put down sink. For powders, centrifuge and decant solution; just decant solution off chunks.
5. You may need to repeat steps 3-4 several times to clear all the fat.
6. Wash samples into plastic centrifuge tubes with water, then centrifuge and discard water.
7. Freeze dry samples
8. Remove from freeze drier, store in dessicator.
Alternate defatting protocol
Chloroform is kind of nasty, so some folks have shifted to dichloromethane/methanol mixtures (2:1 v:v mixtures). If you use this solution, you should freeze dry the samples prior to treatment, then ultrasonic the samples in several volumes of the 2:1 mixture. I don't know how this stuff affects plastic, but I'd do it in glass to be on the safe side. Finally, you could defat with petroleum ether either as above (under the fume hood) or using the Accelerated Solvent Extractor in the SIL. Doing this for powders will be tricky.