After measuring protein concentration by the DC (Detergent Compatible) protein assay (Bio-Rad), export protein concentration report through MD Spectramax 250 software that controls the microplate reader. Copy the content in the exported text file and paste it into Excel, and then save as a UTF-8 format text file.

Example protein concentration file.txt

Use MS Excel or a Jupyter notebook to normalize the protein concentration to 50µg and convert the spectrophotometer output file into the following two files in a format suitable for the Biomek NX-S8:

NX-AMBIC.csv

A	B	C	D	E
scrpos	srcwell	destpos	destwell	vol
media	1	DestPlate	1	33.05
media	1	DestPlate	2	26.73
media	1	DestPlate	3	27.96
media	1	DestPlate	4	28.74
media	1	DestPlate	5	22.94
media	1	DestPlate	6	28.07
media	1	DestPlate	7	24.34
media	1	DestPlate	8	28.12
media	1	DestPlate	9	26.64
media	1	DestPlate	10	26.22

NX-AMBIC.csv output table

NX-protein.csv

A	B	C	D	E
scrpos	srcwell	destpos	destwell	vol
SrcPlate	1	DestPlate	1	10.95
SrcPlate	2	DestPlate	2	17.27
SrcPlate	3	DestPlate	3	16.04
SrcPlate	4	DestPlate	4	15.26
SrcPlate	5	DestPlate	5	21.06
SrcPlate	6	DestPlate	6	15.93
SrcPlate	7	DestPlate	7	19.66
SrcPlate	8	DestPlate	8	15.88
SrcPlate	9	DestPlate	9	17.36
SrcPlate	10	DestPlate	10	17.78

NX-protein.csv output table

Open Biomek software program from Biomek NX-S8 control computer. In the "Instrument" drop-down menu, select "Home all Axes" to prepare the instrument for use and purge air from the tubing and syringes.

Go to "Open Method." Select the "Proteomics" folder and open the method "Proteomics-Normalization Method."

Click on "Instrument Setup."

Set up the deck (refer to the deck setup picture above):

A	B	C
Deck Label	Labware	Reagent
DestPlate	PCR plate 96-well non-skirted (Thermo Fisher, Cat.#AB0600) on a yellow PCR rack
AMBIC	Biomek Reservoir (discontinued)	Ammonium Bicarbonate buffer
tips	200 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#919-262 )

Deck materials

Click on "Transfer From File."

Copy the NX-AMBIC.csv file generated in Excel or via a Jupyter Notebook into the directory that the method is designated to read. For example *C:\Users\jbei\Desktop\Proteomics Methods\CSV files*

Click on the 2nd "View Datasets" to check that you have copy and pasted the correct volumes in the 96-well format.

Click "Finish" to make sure there are no error messages.

Click the "Run" button (green arrow) to start.

Go to "Open Method." Select the "Proteomics" folder and open the method "Proteomics-Plate to Plate Transfer."

Click on "Instrument Setup."

Set up the deck (refer to the deck setup picture above):

A	B	C
Deck Label	Labware	Reagent
DestPlate	PCR plate 96-well non-skirted (Thermo Fisher, Cat.#AB0600) on a yellow PCR rack	Ammonium bicarbonate buffer
SrcPlate	PCR plate 96-well non-skirted (Thermo Fisher, Cat.#AB0600) on a yellow PCR rack	protein
tips	20 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#918-262 ) or
	200 uL pipet tips (Molecular Bioproducts BioRobotix, Cat.#919-262 )

Deck materials

Click on "Transfer From File."

Copy the NX-protein.csv file generated by Excel or via a Jupyter Notebook into the directory that the method is designated to read. For example: *C:\Users\jbei\Desktop\Proteomics Methods\CSV files*

Click on "View Datasets" to check that you have copy and pasted the correct volumes in the 96-well format.

Click "Finish" to make sure there are no error messages.

MANUAL STEP: Use a multichannel pipette to mix protein samples completely right before starting.

Click the Run button (green arrow) to start.

Chemicals to prepare:

• Prepare 100millimolar (mM) by dissolving 28.7mg in 1mL

• Prepare 200millimolar (mM) by dissolving 36.8mg in 1mL

• Prepare 1mg/mL by adding 1mL to 1mg then vortex to mix

Add the following reagents to the normalized protein plate, in this order:

1. 2.5µL
2. 2.5µL
3. 1µL

Incubate at 37°C for 4h 0m 0s to 16h 0m 0s.

After digestion, spin at 14000rpm .

Pipet supernatant into new PCR plate. Seal and store at -20°C until ready for LC-MS/MS analysis (e.g., Discovery Proteomics protocol, Targeted Proteomics protocol).