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 18, 2023
Protocol Integer ID: 55810
Keywords: Proteomics, Sample preparation, Bacteria, Fungi, Protein extraction
Funders Acknowledgement:
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:
- 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
Start with 10 µL of cells per well a non-skirted PCR plate (Thermo Scientific, Cat.#AB0600).
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.
5m
Neutralization and Benzonase treatment
Neutralization and Benzonase treatment
8m
Add 2.75 µL1 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.
3m
Salt-Acetone protein precipitation
Salt-Acetone protein precipitation
8m
Add 200 µL 100% Acetone (Sigma Cat.#179124) to each well and let sit at room temperature for 5 minutes.
5m
Centrifuge at 4000 rpm, 00:02:00 .
2m
Remove supernatant.
1m
Wash and resuspend protein
Wash and resuspend protein
8m
Wash protein pellet twice using 150 µL 80% Acetone (Sigma Cat.#179124).
6m
Add 60 µL of 100 millimolar (mM) Ammonium bicarbonate in 10% Methanol to each well to resuspend protein pellet.