S-adenosylmethionine (SAM) ELISA

Quantify S-adenosylmethionine, the methyl donor for DNA methylation, in coral host tissue by ELISA (BioVision SAM ELISA kit).

Version 1.0 · last updated 2026-10-05 · Used in 1 notebook entries ↓

At a glance

Input	Frozen airbrushed homogenate (host fraction, ~400 µL)
Output	SAM concentration (µg mL⁻¹), normalized to protein or surface area
Kit	BioVision S-Adenosylmethionine (SAM) ELISA kit (manual); Thermo Halt protease inhibitor cocktail
Capacity	One 96-well plate = 8 standards + 40 samples, in duplicate
Time	~1 h sample prep + ~2.5 h assay (estimate)
Tested on	Astrangia poculata

Materials

• SAM ELISA kit: pre-coated plate, SAM standard, Standard/Sample dilution buffer, biotin detection antibody + diluent, HRP-streptavidin (SABC) + diluent, wash buffer concentrate, TMB substrate, stop solution, plate sealers
• Protease inhibitor cocktail (Thermo 78430)
• Refrigerated centrifuge, 37 °C incubator, thermocycler or heat block at 37 °C, −80 °C freezer
• Multichannel pipette, plate reader (450 nm)

Solutions to prepare (per full plate)

Solution	Recipe
Biotin antibody working solution (1:100)	50 µL biotin antibody + 4.95 mL antibody diluent (≥ 4.8 mL needed)
SABC working solution (1:100)	100 µL SABC + 9.9 mL SABC diluent (≥ 9.6 mL needed)
1× wash buffer	30 mL concentrate + 720 mL DI water. Warm to 40 °C if crystals have formed, then cool to room temp
SAM standards	Reconstitute the standard in 1 mL Standard/Sample diluent (= 25 µg mL⁻¹), let stand 10 min, mix gently. Make 2-fold serial dilutions (e.g. 0.3 mL standard + 0.3 mL diluent): 25, 12.5, 6.25, 3.125, 1.56, 0.781, 0.39, 0 µg mL⁻¹. Use within 2 h

Procedure

A. Sample preparation

1. Thaw homogenates on ice.
2. Pellet the symbionts: 6,000 × g, 2 min, 4 °C. Move 400 µL of supernatant (host) to a new tube.
3. Add protease inhibitor at 1:100 (4 µL to 400 µL) and vortex.
4. Lyse with 5 freeze–thaw cycles: 5 min at −80 °C → 5 min at 37 °C.
5. Spin 5,000 × g, 5 min to remove debris. Use the supernatant.

B. ELISA

6. Bring all reagents, standards and samples to room temperature (30 min).
7. Wash the plate 2× with 1× wash buffer.
8. Add 50 µL standard or sample per well, then immediately 50 µL biotin antibody working solution. Seal, tap gently to mix, and incubate 45 min at 37 °C.
9. Discard and wash 3×: fill each well with 350 µL wash buffer, soak 1–2 min, aspirate. After the last wash, blot the plate upside down on paper towels.
10. Add 100 µL SABC working solution per well. Seal and incubate 30 min at 37 °C.
11. Discard and wash 5× as in step 9.
12. Add 90 µL TMB per well. Seal and incubate 15–30 min at 37 °C in the dark, until the first 3–4 standards are visibly blue.
13. Add 50 µL stop solution. Read at 450 nm within 20 min.

C. Calculate

14. Sample and detection antibody go in together, so this is a competitive format: signal decreases as SAM increases (confirm in the kit manual). Fit a 4-parameter logistic curve to the standards and interpolate the samples. Example script: SAM.R.
15. Normalize to protein (from the same homogenate) or to surface area.

Troubleshooting

Problem	Likely cause	Fix
High background in all wells	Incomplete washing	Soak 1–2 min per wash and blot thoroughly after the last wash
Not enough wells for all samples	One plate = 40 samples in duplicate	Choose samples to span treatments and colour range before starting (as in 2019-05-15)
Standards don’t develop	TMB too cold or incubation too short	Check the blue gradient at 15 min and extend to 30 min if needed

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Rewritten from the 2019 Astrangia nutrition run
2019-05	–	First run, 39 A. poculata samples