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Opentrons Script – Proteomics Sample Prep (SDS → SP3 Cleanup)

This is a full automated protocol for prepping proteomics samples on an Opentrons OT-2, from lysis all the way to cleanup. It’s built around SDS-based denaturation, DTT/IAA reduction & alkylation, trypsin digestion, and SP3 bead-based peptide cleanup.

Designed it for 12 samples (we are limited by centrifuge spots, but it’s easy to scale.

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🔧 What It Does:

• 🧼 SDS-based lysis
• 🔥 Protein denaturation with urea buffer
• ✂️ Reduction (DTT) and alkylation (IAA)
• 🍽️ Trypsin digestion
• 🧲 SP3 cleanup w ethanol washes, and elution
• 🧪 Final peptide resuspension

Includes pause points for centrifuge steps, magnet engagement, and SpeedVac drying.

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🧪 Hardware + Labware

• Pipettes:

  • p300_single_gen2 (L)
  • p20_single_gen2 (R)

• Modules:

  • Temperature Module GEN2 (used for 50°C and 37°C incubations)

• Labware:

  • NEST 96-well 100 uL PCR plate (on temp deck)
  • 24-well aluminum block (1.5 mL snapcap tubes)
  • 20 µL and 300 uL tip racks

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💧 Reagents + Liquid Setup

Well	Reagent
A1	SDS Lysis Buffer
A2	SP3 Beads
A3	80% Ethanol Wash Buffer
A4	Elution Buffer (0.1% FA)
A5	Denaturing Buffer
A6	DTT
B1	IAA
B2	Trypsin
B3	Formic Acid (Quench)

Each is pre-loaded using load_liquid() so they appear properly in the App.

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⚠️ Notes

• This protocol is not connected to a magnet module — you'll need to manually place the plate on a magnet and pause at binding/washing steps.
• You can extend this script to include quantification plate transfer or peptide normalization as needed.
• Use as a template and adapt it to your sample type and throughput.

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