TITLE: Bone and tooth collagen extraction for stable isotope analysis and radiocarbon dating
AUTHORS: Prudence Robert, Mathieu Boudin, Samuel Bodé

[DESCRIPTION]
Several collagen extraction protocols are described in the literature, but most lack detailed descriptions of the laboratory manipulations and the specific material used. 
This collagen extraction protocol described here largely follows Longin's (1971) with a few changes. Shortly; The full bone is demineralised using diluted HCl (0.2 to 0.5M). This is followed by removing humic acids using a short NaOH rinse, and by solubilisation and filtration of the collagen.
This protocol aims to provide collagen of appropriate quality for both stable isotope analysis and 14C dating, at a low cost and with a decent duration. This protocol was found to also be suitable, with minor adaptations, to badly preserved samples. 

[BEFORE_START]
400 mg of dry bone sample or half a tooth root (app. 200 mg) is usually sufficient to obtain enough collagen for 14C dating and carbon, nitrogen and sulphur isotope analysis. However, the quantity and quality of the collagen obtained at the end of this extraction protocol remain largely sample-dependent.

[GUIDELINES]
We suggest reading carefully the guidelines and every step of the protocol before starting because some steps and equipment may have to be adapted to your samples. 

Numerous bone and dentine collagen extraction protocols are readily available, but they often lack detailed instructions regarding the process and the material needed. This protocol is tailored for researchers willing to implement stable isotope analysis on skeletal material but, for whom the significant amount of collagen extraction protocols published and tested make their pre-treatment choice uneasy. It can be adapted to fragile archaeological remains (e.g. poorly preserved skeletal material or fish bones).
The protocol has been designed to consider multiple factors simultaneously :
Ensuring a reliable and satisfactory collagen purity suitable for both 14C dating and stable isotope analysis. Discussions with experts such as Mathieu Boudin from The Royal Institute of Belgium (KIK-IRPA) have influenced this aspect, drawing inspiration from Wojcieszak et al. (2020).
Providing a minimally destructive and flexible pre-treatment approach, considering the condition, mass and age of the samples (e.g. Reducing the HCl concentration and temperature used during the demineralisation of highly degraded samples or lightweight samples).  
Emphasising affordability by utilising inexpensive materials and equipment, making the protocol accessible to researchers with limited budgets.
Assuring a relatively short duration for the extraction process.

[STEPS]
SECTION: Removal of lipids and humic acids
14. Remove any DI-water from the tubes with the samples. If the sample was frozen, wait for it to thaw and remove the excess of DI-water. 
SECTION: Demineralisation
10. In a fume hood, add approximately 10 mL of the diluted HCl solution to the reaction tube, ensuring the sample is fully submerged. For well-preserved samples, a 0.5M HCl solution at room temperature can be used.
If the sample seems fragile or highly degraded, consider working at low temperatures (e.g. 4 °C ) and/or with a more dilute acid solution (e.g. 0.2M HCl solution).
Do not close the tubes tightly as CO2  forms during the demineralisation.
 

SECTION: Demineralisation
9. Insert the bone or dentin sample in a reaction tube with a screw cap*.

SECTION: Demineralisation
11. Every two to three days, replace the HCl solution in the tube with fresh HCl. If no bubbling is observed within 30 minutes after the addition of fresh HCl at room temperature (60 min if HCl is stored at 4 °C ), the sample can be considered as demineralised.

 
 

SECTION: Demineralisation
12. Once the skeletal material is demineralised (i.e. no bubbling), rinse it in the tube three times with DI-water.  Rinse also the edges of the tube. If the sample is fragmented, use an Ezee filter.
 

SECTION: Demineralisation
13. If the NaOH step is not directly following, the rinsed demineralised sample can be preserved in the fridge in DI- water. If the sample is crumbly after the demineralisation, it is also possible to remove the DI-water and let the sample sit in the freezer.
 

SECTION: Sample acquisition and cleaning
1. Place the skeletal material to sample on a clean sheet of aluminium foil in a well-ventilated space.
SECTION: Sample acquisition and cleaning
2. With a clean mini diamond saw mounted on a rotary multifunction tool (e.g. Dremel 3000 or Proxxon), carefully cut a piece of the skeletal material aiming for the appropriate mass. The bone powder created during this step can be kept in the foil for further analysis of the material.   
SECTION: Sample acquisition and cleaning
3. With a diamond drill bit, mechanically clean the bone/tooth surface by removing approximately 2mm of its outer surface. 
 

SECTION: Sample acquisition and cleaning
5. Place the samples in a small container. Label the container with the sample ID on several surfaces, and then add DI-water. Sonicate the samples in the container for approximately 3 minutes, avoiding temperature increase. The water may become murky. 
 

SECTION: Sample acquisition and cleaning
7. Let the samples dry  in the container placed in a fume hood.

SECTION: Sample acquisition and cleaning
8. Weight the sample once it is dry.
SECTION: Removal of lipids and humic acids
15. Add 0.25 M NaOH solution making sure to cover the sample. 
SECTION: Removal of lipids and humic acids
16. Let the sample in contact with the NaOH solution for 15 min. Discard the NaOH solution from the tube using a Paster pipette or an Ezee filter if the sample consists of small fragments.
 

 


SECTION: Removal of lipids and humic acids
17. Rinse the sample and the edges of the tube 3 times with DI-water.
SECTION: Neutralising the NaOH
18. Add the diluted solution of HCl (same concentration as for the demineralisation step). Make sure the solution covers the sample. 
 


SECTION: Neutralising the NaOH
19. Let the sample and the solution react for 5 min.

SECTION: Neutralising the NaOH
20. Rinse the sample three times with DI-water. 
SECTION: Solubilisation of the collagen
23. Heat the sample in pH 3 HCL solution at 70 °C  for  2880 min.
 




SECTION: Solubilisation of the collagen
21. Add the pH 3 HCl solution to the tubes. Make sure it largely covers the samples.
 
. 
SECTION: Solubilisation of the collagen
22. Transfer the tubes to a heat-resistant rack.
SECTION: Solubilisation of the collagen
24. While waiting for the samples to solubilise, label the glass vials for each sample and weigh them. Also, prepare small pieces of parafilm to cover each glass vial.
SECTION: Filtration
25. Once the samples are solubilised, filter the liquid samples by slowly pressing the Ezee filters into the sample tubes. Then transfer the filtered liquid to the pre-weighed glass vials designated for each sample.
 



SECTION: Freezing
28. Put a cap above the parafilm and place the glass vials with the filtered samples in a freezer overnight.
SECTION: Filtration
27. Add a piece of parafilm on top of each glass vial and pierce it several times with a sharp tool (e.g. a clean toothpick for each sample).
SECTION: Lyophilisation
29. Remove the caps from the vials and place the frozen samples in a freeze-dryer for at least 24 hours. 
 

SECTION: Weighing and storing
30. Once you obtained fully dry collagen, remove the samples from the freeze-drier.
 


 


SECTION: Weighing and storing
31. After removing the parafilm, place the caps on the glass vials to prevent the collagen from taking up humidity.


 

SECTION: Weighing and storing
32. Weight the dry collagen in the glass vials and calculate the collagen yield.
 

SECTION: Sample acquisition and cleaning
4. The saws and drill bits can be cleaned by being soaked in an ultrasonic bath in DI-water, then in ethanol, then in water again (Repeat until the water is clear), 5 minutes each time.
SECTION: Storage
33. The collagen can be stored in a constantly dry environment (e.g. a freezer).
SECTION: Sample acquisition and cleaning
6. Remove the water from the container, rinse the samples 1 to 3 times and add fresh DI-water. Repeat  until the water in the container remains clear. In general, two sonification steps suffice. 

SECTION: Filtration
26. After the initial filtration, some of the collagen solution (pH 3 HCl solution) may remain at the bottom of the tube. To maximise recovery, add more of the pH 3 HCl solution to the tube. Then, proceed to     to filter the solution again.