Biomineral Protein Extraction Workflow, F. Prada & J. Drake, updated Dec 2025

All steps carried out in a laminar flow biological hood with all preparation tools and surfaces bleached to avoid contamination. Use head net. Beakers, flasks and bottles need to be acid washed and autoclaved before use.

CLEANING FOR CORALS AND FORAMS

MATERIALS

1. Acid-cleaned glass bottle with lid, 500 ml for bleach-hydrogen peroxide mix
2. Acid-cleaned graduated cylinder, 100-250 ml, glass or plastic, for measuring bleach & hydrogen peroxide
3. Commercial bleach
4. Hydrogen peroxide, 30%
5. Ultra-pure water (not DI)
6. Sharpies
7. Conical vials, 50 ml, two per coral specimen (any brand or generic is fine)
8. Vial racks
9. Hammer
10. Balance
11. Transfer pipette, 3 ml
12. Mortar and pestle set, one per person
13. 150 μm pore size sieve, can be shared among multiple users
14. Weigh boats
15. Kim wipes
16. Plastic squirt bottle with 10% bleach solution (can be approximate)
17. Plastic squirt bottle with 1 M HCl
18. Drying oven

LITHS PREPARATION FOR CLEANING

1. After harvesting, centrifuge obtained cells/liths 5,000 x g for 30 mins at RT. Discard supernatant
2. PERCOLL PROTOCOL
3. Re-suspend n 25 mL KNO3, incubate for 20 minutes on shaker at RT.
4. Centrifuge 10,000 x g for 10 minutes. Discard supernatant
5. Repeat 1 and 2 twice in total
6. Re-suspend in 20 mL PERCOLL, incubate for 20 minutes on shaker at RT.
7. Centrifuge 10,000 x g for 10 minutes. Discard supernatant
8. Repeat 3 and 4 six times in total.
9. Re-suspend in filtered (0.2 µm) seawater. Vortex.
10. Centrifuge 10,000 x g, 10 min. Discard supernatant.
11. Repeat 5 and 6 twice in total
12. Using sterile Falcon tubes (only original with blue cap because tubes from other brands contain polymers that leach during decalcification and interfere with enzymatic digestion before sequencing), oxidize in 1:1 of 30% H2O2: 3% NaClO solution using dripper while stirring for 24 hr. Replace solution 1/day (centrifuge at 10,000 x g for 5 minutes at 4°C, remove supernatant and replace with fresh oxidizing solution. (REPEAT 3 TIMES)
13. Wash in ultra-pure water with shaking for 5 minutes, centrifuge at 10,000 x g for 5 minutes at 4 °C, decant and discard rinse water (REPEAT 3 TIMES).
14. Dry powder at 60 °C overnight

CLEANING CORAL FRAGMENTS

1. Bleach, hydrogen peroxide, and HCl are corrosive. PPE (gloves, goggles, and lab coat) should be worn for all steps.
2. Pre-label all tubes with the date of initial (fragments) or second (powder) oxidation, species name and any specimen ID. When completing oxidation steps, add the name or initials of person completing the task.
3. Coral skeleton fragments can be broken into smaller chunks (~1cm2) by wrapping in a Kim wipe and gently hitting with a hammer. Fragments of each specimen should add up to at least 2 g.
4. Oxidize fragments/shells in 1:1 of 30% H2O2: 3% NaClO solution (when making it, leave cap loose) for 30 minutes (3-5 times repeated), in a 50-ml conical vial with a loosened cap. Used solution can be discarded in the sink drain with running water.
5. Wash fragments/shells five times with ultra-pure water for one minute each time and decant. Use a transfer pipette to remove any remaining water droplets in tube base. Decanted rinsate can be discarded in the sink drain.
6. Dry at max 50 °C overnight with a loosened cap or until fully dry (may take a few days).
7. Squirt 10% bleach onto mortar, pestle, and bench area where you will be grinding. Wipe dry with a fresh Kim wipe.
8. Crush cleaned fragments/shells to ≤ 150 μm diameter with a mortar and pestle. Place sieve over weigh boat to capture powder that passes through; re-grind pieces that remain >150 μm. Ground powder should be transferred to a new 50-ml conical vial.
9. When processing multiple samples, rinse mortar, pestle and sieve with MilliQ and then 0.5-1 M HCL, rinse again with MilliQ and wipe with Kim-Wipes between samples.
10. Using 50 mL tubes, oxidize skeleton powder in 1:1 of 30% H2O2: 3% NaClO solution for 30 minutes (3-5 times repeated), with a loosened cap. Centrifuge at 3,000 x g for 3 minutes at 4 °C, remove supernatant and replace with fresh oxidizing solution.
11. Wash in ultra-pure water (Volume of MilliQ should be twice the volume of the powder) while shaking for 5 minutes. Centrifuge at 3,000 × g for 3 min at 4 °C, decant and discard rinse water (REPEAT 3 TIMES).
12. Dry powder at 50 °C overnight with a loosened cap. The next morning, tighten the cap and tap the conical vial several times to dislodge the skeleton. Dry skeleton will easily break apart while wet skeleton will stay clumped and should continue to be dried with a loosened cap.
13. THE FOLLOWING STEPS WERE THE SAME FOR ALL THREE ORGANISMS

CHECK OF CLEANING PROTOCOL

1. Weigh powder (so we know the exact starting weight of the sample and we can then calculate how much protein per g of powder we have)
2. Add 3 volumes of filtered sterile 1X phosphate buffered saline (PBS) to the powder.
3. Vortex for 5 seconds.
4. Sonicate at RT for 30 minutes in a sonication bath with ice to avoid overheating.
5. Centrifuge at 5,000 x g for 10 minutes at 4 C.
6. Transfer supernatant (PBS containing organic matter) into 3 kD ultra filtration tube. Centrifuge filtration tube at 4 °C according to manufacturer instructions, in our case at 5,000 × g, at 4 °C, to concentrate proteins.

DECALCIFICATION WITH DIALYSIS

1. In the laminar flow hood, pour cleaned powdered samples (1-2.5 g) into a 16 cm-long osmotic tube for dialysis (MWCO = 3.5 kDa Thermo Scientific) with 5 mL of 0.2 µm filtered milli-Q water.
2. Place sealed tube into 2 L of 0.1 M CH3COOH (glacial acetic acid) solution under stirring for at least 72 h.
3. The tube containing the dissolved OM is dialysed against 0.2 µm filtered milli-Q water until the final pH is about 6.
4. The obtained aqueous solution containing the OM is poured into a Falcon (needs to be Falcon as other brands tend to leak inhibitory polymers) tube and centrifuged at 5,000 x g for 60 min at 4 °C to separate the soluble (SOM) and the insoluble (IOM) OM fractions.

DECALCIFICATION WITH TUBES

MATERIALS

1. Plastic squirt bottle with 10% bleach
2. Kimwipes or paper towels
3. 0.5 M CH₃COOH (glacial acetic acid) solution in an acid-cleaned glass bottle
4. Cleaned skeleton powder
5. Falcon tubes (usually 1-3 per sample) for collecting SOM solution
6. Conical vial racks
7. Vortexer with sponge-plate for multiple 50-ml conical vials
8. Sharpies
9. Refrigerator
10. Centrifuge filtration (e.g., Amicon or Macrosep) tubes, 3-10 kDa
11. 1.5 ml or 0.6 ml Microcentrifuge tubes
12. Ultra-pure water 13 .Sterile filtered PBS
13. 1 ml and 200 ul pipettes
14. Pipette tips for 1 ml and 200 ul pipettes

METHOD

1. Wipe the exterior of all vessels and equipment that will be handled in the laminar flow hood with 10% bleach and wipe dry with a Kim wipe or paper towel.
2. In the laminar flow hood, pour cleaned powdered samples (1-2.5 g) into a 50 ml Falcon tube (needs to be Falcon as other brands tend to leak inhibitory polymers). Document both the tare weight of the Falcon tube and the weight of the powder+tube. Final protein amounts will be standardized to the dry powder weight.
3. Add 20 ml 0.5 M CH3COOH (glacial acetic acid; HAC) solution to the powder. Swirl gently to mix and liberate initial CO2 and cover all powder grains in acid solution.
4. Allow initial off-gassing with the tube lid loosely fitting for 5 minutes. Then tighten the cap, firmly wedge the tube into the vortexer sponge plate, and shake vigorously (setting 1-2 seems to be good) for ~12 hours at room temperature. Skeletal powder should be full resuspended by the shaking.
5. After turning off the vortexer, gently vent any released CO2 and then reseal the cap.
6. Centrifuge at 5,000 x g for 5 min at 4 °C to separate the soluble (SOM) and the undissolved skeleton.
7. In the laminar flow hood, decant the supernatant containing the SOM into a fresh Falcon tube labeled for the sample. Store the SOM at 4C. Centrifuge filtration (e.g., on an Amicon or Macrosep) can begin immediately or the SOM can be stored until all is accumulated.
8. Add 20 ml fresh 0.5 M glacial acetic acid solution to the undissolved pellet. Repeat the off-gassing, shaking, centrifugation, and decanting until no more pellet seems to dissolve, suggesting that it is only ISOM.
9. Add 1-2 ml fresh 0.5 M glacial acetic acid solution to the remaining ISOM pellet and shake as above for 30-60 minutes. Repeat the centrifugation process as above. Carefully pipette or decant the remaining acetic acid solution into the SSOM pool.
10. Rinse the ISOM pellet in the Falcon tube with 1-3 ml sterile/filtered 1X PBS. Vortex PBS plus ISOM pellet for 10 seconds, then centrifuge at 5,000 x g for 5 min at 4 °C. Decant or pipette PBS supernatant into the SSOM pool and store at 4 °C.
11. SOM concentration by centrifugation on an 3 kDa Amicon/Macrosep should be done at 4 °C at or below the manufacturer’s recommended maximum rotational speed. Ensure proper alignment of the filtration portion of the centrifugation tube if using a fixed angle rotor. Concentration usually takes 30-60 minutes per 20-ml volume. The same Amicon can be used throughout for up to 100 ml of acetic acid-SOM mix; just add more SSOM solution to the top fraction after removing the passed-through fraction from the bottom. Solution that passes through the filter should be saved in a fresh Falcon tube at 4 oC until BCA confirms protein in the concentrated fraction and not in the flow-through.
12. If centrifuge filtration takes more than one work-day, concentrate the acetic acid-SSOM solution to <0.5 ml and then add at least 5 ml sterile 1X PBS to the Amicon. Store overnight at 4 oC. Add more acetic acid-SSOM solution to the top fraction the next day and re-commence centrifuge filtration.
13. Once all SOM has been concentrated, add ~5 ml sterile 1X PBS to the Amicon and centrifuge to wash out the acid. Repeat.
14. Use 200 ul pipette to transfer concentrated and PBS-rinsed SOM to a microcentrifuge tube and add ISOM so we have a combined sample. Note final volume for protein concentration calculations.
15. Aliquot SSOM and ISOM for BCA (~50 uL) and SDS-PAGE (~100 uL) and store at –80C.