Jan 17, 2023
Version 1
Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format V.1
Forked from a private protocol
Peer-reviewed method
- 1Lawrence Berkeley National Laboratory
- LBNL omics
- PLOS ONE Lab Protocols
Protocol Citation: Yan Chen, Jennifer Gin, Christopher J Petzold 2023. Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr6xjpvmk/v1
Manuscript citation:
Chen Y, Gin JW, Wang Y, de Raad M, Tan S, Hillson NJ, et al. (2023) Alkaline-SDS cell lysis of microbes with acetone protein precipitation for proteomic sample preparation in 96-well plate format. PLoS ONE 18(7): e0288102. https://doi.org/10.1371/journal.pone.0288102
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: December 09, 2021
Last Modified: January 17, 2023
Protocol Integer ID: 55810
Keywords: Proteomics, Sample preparation, Bacteria, Fungi, Protein extraction
Funders Acknowledgements:
Dept. of Energy (BER)
Grant ID: Joint BioEnergy Institute (JBEI)
Dept. of Energy (EERE-BETO)
Grant ID: Agile BioFoundry (ABF)
Dept. of Energy (BER)
Grant ID: Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA)
Abstract
This high-throughput protocol details the steps to extract protein from Gram-negative bacteria, Gram-positive bacteria, or non-filamentous fungi in 96-well plate format for quantitative proteomic workflows. This protocol uses a bench-top automated liquid dispenser but the volumes and times also apply to manual and multi-channel pipetter use. This protocol is designed for lab-based, culture conditions and synthetic community experiments where complex sample matrices are minimized. Additional sample preservation and/or protein extraction methods may be required for environmental samples (e.g., feces, soil) to minimize protein degradation and maintain sample integrity.
This protocol works best as part of a high-throughput proteomic sample preparation workflow with:
and
Guidelines
- All centrifuge steps use an Eppendorf 5810R centrifuge.
- You can use Qiagen Lysis Buffer P2 (Qiagen, Cat. #19052) as the alkaline-SDS cell lysis buffer.
Note
Increase reagent volumes accordingly when processing larger amounts of biomass. This protocol works in larger formats (e.g., 1.7 mL Eppendorf tubes) too.
Materials
PCR Plate 96-well non-skirted Thermo Fisher Scientific Catalog #AB0600
Hydrochloric acid Sigma Catalog #320331
Sodium Hydroxide (200 mM)
1% Sodium Dodecyl Sulfate (SDS)
Benzonase nuclease Millipore Catalog #70746
Acetone Sigma Catalog #179124
LC-MS grade Methanol VWR Scientific, Catalog #BJLC230-2.5
Optional:
- Qiagen Lysis Buffer P2 (Qiagen, Cat.#19052) in place of NaOH and SDS
Safety warnings
Acetone is used in this protocol so please follow the appropriate safety guidelines for handling and disposing of non-halogenated solvents at your institution.
Sodium Hydroxide is a HIGHLY CORROSIVE CHEMICAL and contact can severely irritate and burn the skin and eyes with possible eye damage. Inhaling Sodium Hydroxide can irritate the lungs.
Wear gloves and appropriate PPE for safety and to minimize contamination of samples.
Before start
For this protocol you will need:
- a bench-top automated liquid dispenser (e.g., Formulatrix Mantis) or manual/multi-channel pipetters
- an Eppendorf 5810R centrifuge with S-4-104 rotor or similar centrifuge
Mix at least 3 mL of NaOH/SDS buffer for final concentrations of:
- 200 mM NaOH
- 1% SDS
or use Qiagen Lysis Buffer P2 (Qiagen, Cat.#19052)
Cell lysis
Cell lysis
6m
6m
Start with 10 µL of cells per well a non-skirted PCR plate (Thermo Scientific, Cat.#AB0600).
Plate of cell pellets
Add 25 µL of alkaline-SDS cell lysis buffer (200 mM NaOH, 1% SDS) to each well.
1m
Resuspend the cell pellet in lysis buffer on a plate mixer.
Note
Ensure proper mixing of the cell lysis buffer and the cell pellet.
Plate mixer
Cells resuspended in lysis buffer
5m
Neutralization and Benzonase treatment
Neutralization and Benzonase treatment
8m
8m
Add 2.75 µL 1 Molarity (M) Hydrochloric acid (Sigma Cat.#320331) to each well.
2m
Add 25 µL 100 mM Ammonium bicarbonate (VWR Scientific Cat.#BJ40867-50G) and 0.5 µL Benzonase nuclease (Millipore Cat.#70746) to each well.
3m
Mix thoroughly on the plate mixer.
Cell lysate after benzonase treatment
3m
Salt-Acetone protein precipitation
Salt-Acetone protein precipitation
8m
8m
Add 200 µL 100% Acetone (Sigma Cat.#179124) to each well and let sit at room temperature for 5 minutes.
Protein precipitation after mixing in 100% acetone
5m
Centrifuge at 4000 rpm, 00:02:00 .
2m
Remove supernatant.
Protein pellet after centrifugation and removal of supernatant
1m
Wash and resuspend protein
Wash and resuspend protein
8m
8m
Wash protein pellet twice using 150 µL 80% Acetone (Sigma Cat.#179124) .
Note
Increase the number of washes depending on the sample matrix background.
6m
Add 60 µL of 100 millimolar (mM) Ammonium bicarbonate in 10% Methanol to each well to resuspend protein pellet.
Protein pellet resuspended in 60 uL of Ammonium bicarbonate in 10% Methanol
2m
Store at -20 °C until ready for Automated Protein Quantitation with the Biomek-FX liquid handler system.