Jan 12, 2022

Public workspaceAutomated Protein Quantification with the Biomek-FX Liquid Handler System V.2

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.b3grqjv6
  • 1Lawrence Berkeley National Laboratory
  • LBNL omics
  • PLOS ONE Lab Protocols
Jennifer Gin
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DOI: dx.doi.org/10.17504/protocols.io.b3grqjv6
External link: https://doi.org/10.1371/journal.pone.0264467
Protocol Citation: Yan Chen, Nurgul Kaplan Lease, Tad Ogorzalek, Jennifer Gin, Christopher J Petzold 2022. Automated Protein Quantification with the Biomek-FX Liquid Handler System. protocols.io https://dx.doi.org/10.17504/protocols.io.b3grqjv6Version created by Christopher J Petzold
Manuscript citation:
Chen Y, Lease NK, Gin JW, Ogorzalek TL, Adams PD, Hillson NJ, Petzold CJ (2022) Modular automated bottom-up proteomic sample preparation for high-throughput applications. PLoS ONE 17(2): e0264467. doi: 10.1371/journal.pone.0264467
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: January 04, 2022
Last Modified: January 12, 2022
Protocol Integer ID: 56561
Keywords: Protein quantification, Automation, Biomek, Lowry assay, Proteomics, Sample preparation
Abstract
This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards.

This protocol works best as part of a full proteomic sample preparation workflow with:

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System

and

Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System
Image Attribution
Jonathan Vu
Guidelines
- A Beckman-Coulter Biomek FX liquid handler system with a 96-pod head is used for this protocol. Alternative liquid handlers can be used with appropriate method development.

- A Molecular Devices Spectramax 250 microplate reader is used for the protein quantification assay measurement.

- Because different deck orientations and system components are possible, you will need to modify the method file (attached in the 'Before start' section) for your specific Biomek liquid handler system.

Notes:
- This protocol is set up to measure the amount of protein in duplicate.
Materials
Hard-Shell 96-Well PCR Plates low profile thin wall skirted white/clearBIO-RADCatalog #HSP9601
Pierce Bovine Serum Albumin Standard Pre-Diluted Set Thermo Fisher, Catalog #23208
20 uL pipet tips Molecular Bioproducts BioRobotix, Catalog #918-262
200 uL pipet tips Molecular Bioproducts BioRobotix, Catalog #919-262
Corning 96 Well Black Polystyrene Microplate Fisher Scientific, Catalog #07-200-567
Reservoir Microplate Agilent, Catalog #201254-100
96 Deep Well Reagent Reservoir VWR, Catalog #101100-962
Water LC-MS grade B&J Brand VWR Scientific, Catalog #BJLC365-2.5
DC Protein Assay Reagent A by Bio-rad Laboratories, Catalog #500-0113
DC Protein Assay Reagent B by Bio-rad Laboratories, Catalog #500-0114
8-strip PCR Tubes with Caps Axygen, Catalog #14-222-251
Safety warnings
Wear proper PPE (gloves, safety goggles, and lab coat), and prepare solvents in a chemical fume hood.
Store organic solvents in a flammable storage cabinet when not in use.
Discard used solvents and buffers in appropriate waste containers.
Before start
Prepare BSA Standards Plate (1st 4 rows from A to D):
  1. Add 40 uL of H2O into wells A1 to D1.
  2. Add 40 uL of BSA Standards 1 (125 ug/mL) to 7 (2000 ug/mL) into columns 2 to 8.
For this protocol you will need:
  1. Beckman-Coulter Biomek FX liquid handler system with a 96-pod head.
  2. Upload the attached method file and modify it to fit your deck and system configuration.
Download Modular Protein Quantitation method.bmfModular Protein Quantitation method.bmf

Deck Setup
Deck Setup
10m
10m
Open Biomek Software that controls Biomek-FX liquid handler system. Under "File" drop-down menu, click "Open" to select the automation method "Modular Protein Quantitation method."

Note
Because different deck orientations and system components are possible, you will need to modify the method file (attached in the 'Before start' section) for your specific Biomek liquid handler system.

Click on "Instrument Setup" under the "Setup" group node to get visual instruction on how to set up the deck.
Set up the deck (refer to the deck setup picture below):

ABC
Deck LabelLabwareReagent
proteinPCR96 plate (BIO-RAD, Cat.#HSP9601)unknown amount of protein to quantify
titratePCR96 plate (BIO-RAD, Cat.#HSP9601)
BSA standardsPCR96 plate BSA Standards (Thermo Fisher, Cat.#23208)
tips1,220 µl pipet tips (Molecular Bioproducts BioRobotix, Cat.#918-262 )
tips 3,5200 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#919-262 )
control read, prot read 1, prot read 2Black Microplate (Fisher Scientific, Cat.#07-200-567)
Buffer AReservoir Microplate (Agilent, Cat.#201254-100)DC Protein Assay Reagent A (Bio-rad Laboratories, Cat.#500-0113)
waterReservoir Microplate (Agilent, Cat.#201254-100)LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5)
Buffer B96 Deep Well Reagent Reservoir (VWR, Cat.#101100-962) DC Protein Assay Reagent B (Bio-rad Laboratories, Cat.#500-0114)
Materials for Deck setup


Deck setup
Labware for Deck setup
10m
MANUAL STEP: Use a multichannel pipette to mix protein samples right before starting.
2m
Click the "Run" button (green arrow) to start.
DC protein assay
DC protein assay
25m
25m
Transfer 25 µl of Buffer A to Protein Read plate 1, Protein Read plate 2, and Control Read plate.



1m
Transfer 12 µl of H2O into Titrate plate. Then transfer 3 µl (see Note for more details) from Protein plate to Titrate plate and mix with 5 cycles of pipetting mixing on deck.

Note
Note: The dilution factor could be altered as needed by changing the volumes of water and proteins transferred to the titration plate. Be sure to multiply the protein quant results by the dilution factor before you do your normalization calculation.



1m
Transfer 5 µl of protein from Titrate plate to Protein Read plate 1 and Protein Read plate 2.



1m
Transfer 5 µl from BSA Standards plate to Control Read plate.



Note
Prepare BSA Standards plate (1st 4 rows from A to D):
Add 40 µl of H2O into wells A1 to D1.
Add 40 µl of BSA Standards 1 (125 µg/ml) to 7 (2000 µg/ml) into columns 2 to 8.

1m
Method will pause until user resumes it again. Set a timer for 5 minutes.


5m
After 5 min, click OK to resume method.
Transfer 200 µl from Buffer B to the 3 Read plates and incubate for Duration00:10:00 .

The method will pause until user resumes it again. Set up a 10 minutes timer and click OK afterwards to finish.



10m
Spectrophotometer reading for protein Quantification
Spectrophotometer reading for protein Quantification
10m
10m
Transfer plates to the microplate reader (MD Spectramax 250) to read absorbance at 750 nm and calculate protein concentration.


1m
Read Control Read plate.


ABC
SampleConcentrationMean OD Value (Absorbance)
St011250.024
St022500.042
St035000.090
St047500.138
St0510000.165
St0615000.239
St0720000.281
Standards (µg/ml)


Example Standards Curve


1m
Read Protein Read plate 1 and Protein Read plate 2.


AB
SampleConcentration
Un88585.249
Un89785.257
Un90670.135
Un91718.864
Un92868.962
Un93679.907
Un94743.064
Un95994.173
Un961115.072
Examples of Sample concentrations that are reported by MD Spectramax 250
Note
Remember to multiply the protein concentrations by five (5) to account for the five-fold dilution in Step #7.

2m