TITLE: Automated 96 well plate based protein quantitation using a Beckman Biomek™ NxP workstation and a Pierce™ 660nm Protein Assay Kit
AUTHORS: ronan.ocualain

[DESCRIPTION]
These include 
sample lysis, 
protein extraction, 
solubilisation, 
estimation, 
reduction and alkylation, 
normalisation, 
clean-up, 
enzymatic digestion, 
and desalting. 


Adapting these steps onto an automated workstation can increase efficiency, throughput, and reduce coefficients of variance (%CV) thereby providing reliable reproducible data for statistical comparisons.  


This protocol is part of a modular collection for the processing of biological samples for proteomics. 

[BEFORE_START]
While this step is part of a modular format in the automated process of sample preparation for proteomics, it may also be performed in a stand-alone capacity. In the modular workflow it is usually performed after ultrasonication and reduction and alkylation of lysates.

In this method, the sample concentrations are assayed from either the Covaris LE220+ and either 96 AFA-Tube TPX plate (PN 520291), or 8 AFA -Tube TPX strip (PN 520292).

Other microplates for sample estimation may be used - plate information and location must be updated in the Deck layout and Instrument setup part of the method.

The method may be accessed here:

  


[GUIDELINES]
A Beckman Biomek NxP with Span-8 pod and associated software is used in this method. 
Of course, alternative liquid handlers can be used with appropriate method development.

The Biomek is a versatile liquid handler, but this means that alternative deck orientations and system components are possible. You may need to modify the method file for your specific Biomek liquid handler system.

[STEPS]
SECTION: Deck layout
3. Add the sample plate to be measured in P1 of the deck.
 

SECTION: Deck layout
1. To a Beckman full reservoir, add 50 mL of Pierce 660nm protein assay reagent with 1 g of ionic detergent compatibility reagent (IDCR). This can be prepared by adding the 1g from a packet of IDCR to a 50 mL falcon tube, and adding 50 mL of Pierce 660nm assay reagent, with mixing by inverting. Place the reservoir at P4 of the deck, and label it Assayreagent.

SECTION: Deck layout
2. To the deck of the Biomek NxP, add a box of p250 tips to P8, and two boxes of p20 tips to P2, and P6 respectively. 
SECTION: Deck layout
5. Add a Pierce microplate to P3, and label it read.

SECTION: Deck layout
4. To a Beckman full reservoir, add 50 mL of deionised water, and place in P7 of the deck, label this diluent.
SECTION: Deck layout
6. The deck should look like the following
 

SECTION: Setting up the deck
7. Double click the software icon.
SECTION: Setting up the deck
8. Under the Method tab, select home all axes to orient and prepare the automated liquid handler.
SECTION: Setting up the deck
9. Under File, select Open/Method. Select the ProteinQuantitation660nmAssayV01 method.
SECTION: Setting up the deck
10. To the deck of the NxP, add two p20 tip boxes, and 1 p250 tip box.
The Sample will be in P1 position, while the AssayReagent is at P4. The diluent will be at P7.
    
SECTION: Operating procedure
12. The default liquid sample volume for this method is 120uL.
Volumes may be changed in plate properties, and the dilution ratio of the samples to be measured may be adjusted in the Start tab of the program

 
The dilution in this assay of sample is 1:5, where 2 µL of sample is added to 8 µL of diluent.

SECTION: Running the assay
13. Start the method by clicking the green Run icon.

 

SECTION: Running the assay
14. You will be prompted by the software to enter the location of the first column to be processed.
The method is set up to transfer and dilute columns 1 to 10 of the sample plate to the read plate.
This is because columns 11 and 12 are used for generating the standard curve.
If your first samples are in column A1, enter "1"
You will then be prompted to enter the value of the last column to be processed. If you have a full plate of 80 samples, enter "10". All 80 wells will be processed.
SECTION: Running the assay
15. The software will ask you to check that the deck layout matches that of the program. Once you are satisfied that this is the case, click OK.


The instrument will now dilute, and add assay reagent to your diluted samples.


 


SECTION: Measurement
16. Once shaking of the plate has completed, you may now measure the plate using endpoint absorbance at a wavelength of 660nm to measure the protein concentration of samples from columns 1 to columns 10. Columns 11 to 12 are used to set up the calibration curve.

SECTION: Setting up the deck
11. Add a Read plate to P3 (A Pierce 96 well microplate #15041 in this case).
It is at this step that the calibrants for the generation of the standard curve be added, in columns 11 and 12. I recommended using Thermo Scientific‱ Pierce‱ Bovine Serum Albumin Standard Pre-Diluted Set (23208). These are a set of 7 protein standards ranging from 2 µg/µL to 0.125 µg/µL . The last available pair of wells can be used for the diluent blank.

SECTION: Measurement
17. The results may be exported to an .csv file, and transformed to provide volumes for normalisation of samples prior to processing, clean-up, and digestion.
SECTION: Data transformation
18. Data from the 660nm assay may be edited as below:

 
In the above example, individual protein lysates were measured in wells A1 to H1. From this, the volume of lysate needed to process 10 µg was calculated, along with the amount of HEPES buffer to give a final volume of 25 µL .

SECTION: Data transformation
19. This data may then be used to make a .csv file as detailed below

 

SECTION: Data transformation
20. Once this is complete, modify the file
   
and add the .csv file to it as follows

 
Upload the .csv file prepared earlier in the file name tab.
Note, it is important that the headers match, in this case, these are 
Source, sourcewell, dest, and dest well, along with volume.