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

[DESCRIPTION]
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

[BEFORE_START]
Prepare BSA Standards Plate (1st 4 rows from A to D):
Add 40 uL of H2O into wells A1 to D1.
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:
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.
        


[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. 

[STEPS]
SECTION: Deck Setup
1. 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."

 

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

    

SECTION: DC protein assay
7. 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.  

 


   
SECTION: DC protein assay
8.  Transfer 5 µl of protein from Titrate plate to Protein Read plate 1 and Protein Read plate 2.

   

SECTION: DC protein assay
9. Transfer 5 µl from BSA Standards plate to Control Read plate.

     

 

SECTION: DC protein assay
10. Method will pause until user resumes it again. Set a timer for 5 minutes.


SECTION: DC protein assay
11. After 5 min, click OK to resume method.
SECTION: DC protein assay
12. Transfer 200 µl from Buffer B to the 3 Read plates and incubate for 10 min .

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

   

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


SECTION: Spectrophotometer reading for protein Quantification
14. Read Control Read plate.


 


 
   


SECTION: Spectrophotometer reading for protein Quantification
15. Read Protein Read plate 1 and Protein Read plate 2.


 
 
 

SECTION: Spectrophotometer reading for protein Quantification
16. Store protein plate at -20 °C until ready for Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System.