Total protein (alkaline digestion + Pierce BCA assay)

Solubilize protein from coral homogenates or larvae with NaOH, neutralize, and quantify with the Pierce BCA microplate assay.

Version 1.0 · last updated 2026-10-05 · Used in 5 notebook entries ↓

At a glance

Input	200 µL homogenate (holobiont, host or symbiont fraction) or larval homogenate
Output	Total protein (mg mL⁻¹), normalized per larva or per cm²
Kit	Thermo Scientific Pierce BCA Protein Assay Kit (manual)
Time	4 h digestion + ~1 h assay
Tested on	Porites astreoides adults and larvae; Astrangia poculata

Materials

• Pierce BCA Protein Assay Kit (Reagent A, Reagent B, BSA standard)
• 1 M NaOH, 0.1 M HCl, pH paper
• Thermomixer or shaking incubator at 50 °C
• 96-well flat-bottom plate, plate reader (562 nm), 37 °C incubator

Procedure

A. Alkaline digestion

1. Thaw samples. For fractions, prepare them first (tissue protocol, Part B):
   • Holobiont: 200 µL vortexed homogenate.
   • Host: spin homogenate 15,000 × g, 3 min; use 200 µL of the supernatant.
   • Symbiont: resuspend that pellet in 1 mL 1× PBS; use 200 µL.
2. Add 10 µL 1 M NaOH to each 200 µL sample (pH ~10). Spot a tiny drop on pH paper to check, since the vials are too small for a probe.
3. Incubate 4 h at 50 °C, shaking at 300 rpm. Prepare the BCA standards and working reagent during this time.
4. Neutralize with 80 µL 0.1 M HCl, added in small steps, checking with pH paper until pH ≈ 7. Don’t continue until the pH is 7.
   • 80 µL was enough in the 2021 pH test. The original 2018 protocol used up to 280 µL. Samples differ, so always check the pH.
5. Record the final volume (e.g. 200 + 10 + 80 = 290 µL, a dilution factor of 1.45).

B. BCA microplate assay (from the kit manual)

6. Make BSA standards in the same diluent as your samples (PBS plus the NaOH/HCl), following the kit dilution table.
7. Make working reagent: Reagent A : Reagent B = 50 : 1. You need 200 µL per well.
8. Pipette 25 µL of each standard and sample into the plate, in duplicate or triplicate.
9. Add 200 µL working reagent to each well and mix on a plate shaker for 30 s.
10. Cover and incubate 30 min at 37 °C. Cool to room temperature.
11. Read absorbance at 562 nm.

C. Calculate

12. Subtract the blank, fit the standard curve (the BCA curve is usually slightly non-linear; a quadratic fit is fine), and calculate µg mL⁻¹.
13. Multiply by the dilution factor from step 5, then normalize: × homogenate volume ÷ surface area, or ÷ number of larvae.

Troubleshooting

Problem	Likely cause	Fix
Purple color everywhere, values too high	Sample pH not neutralized, or too much HCl	Re-check the pH; BCA is sensitive to pH and reducing agents
Values below the lowest standard	Sample too dilute	Use more homogenate, or a lower-range standard curve
Replicates disagree	Particulates in the well	Spin samples briefly before pipetting into the plate

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Rewritten from the 2018 protocol; neutralization volume updated from the 2021 pH test; BCA steps and calculation added
2021-06	–	pH test: 80 µL 0.1 M HCl neutralizes 10 µL 1 M NaOH in 200 µL homogenate; host/symbiont/holobiont fractions
2018-10	–	Original alkaline digestion for larval tissue