Feb 26, 2026
Cell lysis for preparation of protein extracts for quantitative proteomics - Kryptoperidinium foliaceum, Nitzschia laevis and Symbiodinium sp.
- Will Lewis1,
- Ross F. Waller1
- 1Department of Biochemistry, University of Cambridge
Protocol Citation: Will Lewis, Ross F. Waller 2026. Cell lysis for preparation of protein extracts for quantitative proteomics - Kryptoperidinium foliaceum, Nitzschia laevis and Symbiodinium sp.. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvry7o2gmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 04, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 242558
Keywords: lysis of dinoflagellate, dinoflagellate, kryptoperidinium foliaceum, cell lysis for preparation, quantitative proteomic, symbiodinium sp, cell lysi, protein extracts for quantitative proteomic, lyse cell, unable to lyse cell, tough cell wall, containing concentrated urea, cell, bead sonication, lysis buffer, concentrated urea
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: https://doi.org/10.37807/GBMF11532
Abstract
This methodology was developed for the lysis of dinoflagellates and diatoms with tough cell walls, using glass-bead sonication in a lysis buffer containing concentrated urea and detergent. This was necessary for strains where sonication without the presence of glass beads was unable to lyse cells.
Guidelines
Always use HPLC-grade water throughout this protocol
Materials
Glass beads, acid-washed <106µm G4646-100G SigmaAldrich
Triton X-100
HPLC-grade water
Urea
1M Triethylammonium bicarbonate (TEAB) buffer
15 mL tubes
Equipment:
Diagenode Bioruptor Plus sonication device
MX-T6-S Classic Roller Mixer with 6 Rollers
biosan Orbital thermo-shaker TS-100C Smart
Troubleshooting
Before start
Turn on the Bioruptor to begin cooling the waterbath to 4°C
Pre-warm Triton X-100 to 37 °C
Preparation of buffers
Ensuring glass beads are well-suspended in a buffer containing Triton X-100 is challenging since the Triton X-100 will cause excessive foaming when vortexing, making pipetting of the buffer challenging.
Therefore this protocol uses two buffers.
Buffer A: Lysis buffer
8M Urea, 1% Triton X-100, 0.1 M TEAB
Buffer B: Beads-in-buffer suspension
8M Urea, 0.1 M TEAB, acid-washed <106 µm glass beads
Prepare Buffer A: Lysis buffer
Make a 10% dilution of Triton X-100 in HPLC grade water (1 volume of Triton X-100 in 9 volumes of water)
For accurate pipetting, Triton X-100 should be pre-warmed to 37 °C to make it less viscous
Per sample this buffer contains:
96 mg Urea
20 µL 1M TEAB
30 µL 10% Triton X-100
With HPLC-grade water added to make a total volume of 200 µL.
However, Urea should be fully dissolved before adding Triton. Therefore use the following protocol steps to prepare the buffer. To make larger volumes for larger numbers of samples, the amounts and volumes of the components have been calculated in the following table.
| A | B | C | D | E | |
| 1 sample | 15 (+2) samples | 20 (+2) samples | For 5 mL | ||
| Urea | 0.096 g | 1.633 g | 2.114 g | 2.402 g | |
| 1M TEAB | 20 µL | 340 µL | 440 µL | 500 µL | |
| 10% Triton X-100 | 30 µL | 510 µL | 660 µL | 0.75 mL | |
| Total volume when water added | 200 µL | 3400 µL | 4400 µL | 5000 µL |
Combine the total amount of Urea and the TEAB in a tube.
Calculate ~60% of the total volume of the buffer being prepared, and then add this volume of HPLC-grade water to the Urea and TEAB. Vortex to dissolve the Urea.
Add the total amount 10% Triton X-100 slowly and gentley to avoid foaming
Add HPLC-grade water to top-up to the total volume of the buffer.
Highly precise ratios are not overly critical, and therefore I typically prepare this buffer in a 15 mL tube and use the measurements printed on the side of the tube as a guide when topping up to the final volume with HPLC-grade water.
Buffer B: Beads-in-buffer suspension
Combine Urea and 1M TEAB, and top up to the total buffer volume with HPLC-grade water according to this table below, depending on the number of samples being processed. Once these first three components have been combined to the right volume, then the corresponding quantity of glass beads can be added. Then place on roller mixer to keep the beads suspended.
| A | B | C | D | E | |
| 1 sample | 15 (+2) samples | 20 (+2) samples | For 5 mL | ||
| Urea | 0.096 g | 1.633 g | 2.114 g | 2.402 g | |
| 1M TEAB | 20 µL | 340 µL | 440 µL | 500 µL | |
| Total volume when water added | 200 µL | 3400 µL | 4400 µL | 5000 µL | |
| Glass beads | 200 mg | 3.4 g | 4.4 g | 5 g |
Processing cell pellets for lysis
Pre-cool a centrifuge with adapter for 1.5 mL and 2 mL tubes to 4 °C
Remove cell pellets in 2 mL tubes from -80 °C freezer and put on ice for this step only until buffers are added.
Add 200 µL Buffer A to each cell pellet sample and then place the tube in a orbital shaker and leave for a few minutes to resuspend. I use a biosan orbital thermo-shakerTS-100C Smart set to maximum speed at room temperature.
Whilst keeping the glass beads in Buffer B suspended and evenly distributed by using a combination of a roller mixer and a vortex, add 200 µL of this beads-in-buffer suspension to each sample.
Place each sample in the bioruptor. Set to the bioruptor to "High" and sonicate for 15 seconds on, 15 seconds off, for 20 cycles (10 minutes total).
Using 200 µL pipette, transfer all the liquid from each sample into a new tube (some beads are likely to be carried over.
Centrifuge at 16 000 x g for 1 minute to pellet beads and cell fragments.
Transfer the supernatant to a new tube and store at -20 °C until ready to process for quantitative proteomics using mass spectrometry.