TITLE: Automated 96 well plate based protein reduction and alkylation using a Beckman Biomek™ NxP workstation 
AUTHORS: ronan.ocualain

[DESCRIPTION]
Sample preparation for mass spectrometry analysis involves numerous liquid transfer steps.

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]
This step should be performed after ultrasonication of samples using the Covaris LE220+ and either 96 AFA-Tube TPX plate (PN 520291), or 8 AFA -Tube TPX strip (PN 520292).

If this is not the case, place the plate containing the protein lysate to be reduced and alkylated in the Biomek workstation. For the method here, this is P1.
Method:   

Upload and open the attached method and modify it to your system and deck configuration.

To prepare:
Prepare 100 millimolar (mM) dithiothreitol (DTT) by adding 92.4 mg to a 15 mL Falcon tube. Add 6 mL of milli-Q H2O, vortex mix. Store for 12 hours, then discard.
Prepare 100 millimolar (mM) iodoacetamide (IAM) by adding 55.5 mg to 15 mL Eppendorf tube. Add 3 mL of milli-Q H2O, vortex mix. IAM is light sensitive, cover with foil. Store for 12 hours, then discard.



[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: Preparation
1. Double click the software icon.
 

SECTION: Preparation
2. Under the Method tab, select home all axes to orient and prepare the automated liquid handler.
SECTION: Preparation
3. Under File, select Open/Method. Select the ProteinLysateReductionAlkylationV01 method.
SECTION: Preparation
4. To the deck of the NxP, add two p20 tip boxes, and 1 p250 tip box.
Add the samples in a plate to be reduced and alkylated. For the above method, this will either be a 96 AFA-Tube TPX plate, or multiples of 8 AFA-Tube TPX strips in a 8 AFA-tube TPX strip holder. If using a plate other than these, enter its details in the instrument set up step.
SECTION: Preparation
5. To a Eppendorf 96/500uL plate (reagent plate), add the following volumes into each of the 8 wells of a single column of an Eppendorf 96/500uL protein lobind deepwell plate.
Column 1: 500 uL of 100mM DTT
Column 2: 250 uL of 100mM IAM
 

SECTION: Setting up the deck
6. Place tip boxes, reagent, and sample plates as detailed below
 
It is important that the deck layout in the software matches that on the instrument.
Mismatches can result in damage to pod.


SECTION: Setting up the deck
7. The default liquid sample volume for this method is 120uL.
Volumes may be changed in plate properties
 
 

SECTION: Starting the method
8. Start the method by clicking the green Run icon.
 

SECTION: Starting the method
9. You will be prompted by the software to enter the location of the first column to be processed.
If your first samples are in column A1 to H1 on your sample plate, 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 96 samples, enter "12". All wells will be processed.

SECTION: Starting the method
10. 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 reduce, alkylate, and quench the samples.

 


SECTION: Starting the method
14. 
After the program has completed, you may now proceed to protein concentration estimation, or store the samples at either -20 °C or -80 °C .

SECTION: Starting the method
13. Do not reach into the instrument while the program is running, this action will break the "light curtain" and stop the system as a safety precaution.
SECTION: Starting the method
11. After the reduction step, the system will pause, and the user will be prompted to remove the plate and incubate it at 60 °C for 15 min 
 

SECTION: Starting the method
12. After the alkylation step, the system will pause, and the user will be prompted to remove the plate, cover it, and incubate it at 60 Room temperature in the dark for 30 min