TITLE: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System
AUTHORS: Yan  Chen, Tad  Ogorzalek, Nurgul  Kaplan Lease, Jennifer  Gin, Christopher J Petzold

[DESCRIPTION]
This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck".

This protocol works best as part of an automated proteomic sample preparation workflow with:

Automated Protein Quantitation with the Biomek-FX liquid handler system

and

Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

[BEFORE_START]
For this protocol you will need:
- a Beckman-Coulter Biomek FX liquid handler system with a 96-pod head
- Upload the attached method file and modify it to fit your deck and system configuration
  
- an Eppendorf 5810R centrifuge with S-4-104 rotor or similar centrifuge




[GUIDELINES]
- A Beckman-Coulter Biomek FX liquid handler system is used for this protocol. Alternative liquid handlers can be used with appropriate method development.

- 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.



[STEPS]
SECTION: Deck Setup
1. Open Biomek Software that controls Biomek-FX liquid handler system. Under "File" drop-down, click "Open" to select the automation method "Modular Protein Extraction method."

 

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


 

   
 
 
 
 
 
 
SECTION: Deck Setup
4. Click the "Run" button (green arrow) to start.
SECTION: Protein Extraction
5. Transfer 87.5 µl of water from water reservoir to cell plate. Mix and resuspend cell pellet with 20 cycles of pipetting mixing on deck. 

 

  

SECTION: Protein Extraction
6. Transfer cell resuspension to PCR96 plate.
  

SECTION: Protein Extraction
7. 
The PAUSE step prompts you to centrifuge your PCR96 plate.
SECTION: Protein Extraction
8. Centrifuge 2000 x g, 2 min, 25 °C .
SECTION: Protein Extraction
9. Put PCR96 plate back on deck space (refer to deck setup picture above) -- PCR96 plate "prot."  Swirl and pour solvent (4:1, Methanol:Chloroform) into reservoir.

 

SECTION: Protein Extraction
10. Remove supernatant by transferring 87 µl from PCR96 plate back to cell plate.
SECTION: Protein Extraction
11. Transfer 125 µl of solvent (4:1, Methanol:Chloroform) to PCR96 plate. Mix to promote cell lysis with 20 cycles of pipetting mixing on deck. 
 

  

SECTION: Protein Extraction
12. Transfer 75 µl of water into PCR96 plate and seal the plate with peelable seal at microplate sealer. Gently mix the samples by inverting the plate 5 times.  
  

SECTION: Protein Extraction
13. The PAUSE step prompts you to centrifuge your PCR96 plate.  

SECTION: Protein Extraction
14. Centrifuge 4000 rpm, 2 min, 25 °C .

 

SECTION: Protein Extraction
15. Put PCR96 plate back on deck space (refer to deck setup picture above) -- PCR96 plate "prot."  
SECTION: Protein Extraction
16. Remove supernatant by transferring 170 µl of top layer from PCR96 plate to cell plate.



SECTION: Protein Extraction
17. Add 75 µl of methanol and mix with 20 cycles of pipetting mixing on deck.

 


  

SECTION: Protein Extraction
18. The PAUSE step prompts you to centrifuge your PCR96 plate.  
 

SECTION: Protein Extraction
19. Centrifuge 4000 rpm, 2 min, 25 °C .
SECTION: Protein Extraction
20. Put PCR96 plate back on deck space (refer to deck setup picture above) -- PCR96 plate "prot."  
SECTION: Protein Extraction
21. Remove supernatant.
SECTION: Protein Extraction
22. Resuspend in 100 µL 100 millimolar (mM) and mix the samples with user defined cycles of pipetting mixing on deck. 
  
 


SECTION: Protein Extraction
23. Store at -20 °C until ready for Automated Protein Quantitation with the Biomek-FX liquid handler system.