Introduction

Challenge
Simple Western™ Jess is a high throughput western blot technique with truly quantifiable data. However, preparing the samples and Simple Western™ plate is time consuming, laborious, and is prone to small errors.

Solution
The CyBio FeliX was used to perform all liquid handling aspects of the sample protein assay and the Simple Western™ plate preparation.

The Simple-Western™ Jess is a capillary based automated western blot instrument capable of running 24 samples from generation to results in approximately 5 hours. This throughput is advantageous compared to traditional western blot systems that traditionally runs 10-20 samples, has 4-5 hours bench time, and takes 24-48 hours to get results.

Automation of the protein separation and immunodetection process eliminates the error prone steps of traditional western blotting and the associated software quickly processes the quantifiable data. At Aurelia Bioscience, the Simple-Western™ Jess is a staple technique in a repertoire of drug discovery methodologies.

For this reason, the CyBio FeliX liquid handling system was used to simplify the Simple-Western™ set-up procedure.

Automation of the liquid handling aspect of Jess plate preparation would further increase the reproducibility of the data, allow increased flexibility of a scientist’s workload by removing the manual aspects of the preparation, and allow inexperienced users to access the technology. To demonstrate the capabilities of CyBio FeliX, THAL-SNS-032, a well-studied Proteolysis-targeting chimera (PROTAC) molecule and specific degrader of cyclin dependent kinase 9 (CDK9) was used to demonstrate the capability of the system and compare the results to previously generated data used for the Protein-Simple™ webinar “Advancing Targeted Protein Degradation Research with Automated Western Blotting”.

Materials and Methods

Samples and reagents
  • Pierce BCA protein assay kit – Thermo Fisher #23227 (Reagents B and A are mixed at a 1:50 ratio prior to use)
    • BCA reagent A, 500 ml
    • BCA reagent B, 25 ml
  • 12-230kDa Jess/Wes separation module 8x25 capillary cartridges – Bio-Techne #SM-W004
  • Anti-Rabbit Detection module for Jess/Wes – Bio-Techne #DM-001
  • Anti-Mouse Secondary NIR antibody – Bio-Techne #043-821
  • MV-4-11 Cells – ATCC #CRL-9591
  • RPMI-1640 – Thermo Fisher #21875-034
  • Fetal Bovine Serum – Corning #35-079-CV
  • PBS tablets – Thermo Fisher 18912014 (1 tablet per 500 mL sterile water)
  • 1x Complete RIPA lysis buffer:
    • RIPA – Thermo Fisher #89900
    • Magnesium Chloride – Merck #M1028-100 ml (FAC 2mM)
    • Benzonase clease – Merck #E1014-25KU (0.5 µL per mL)
    • Pierce Protease Inhibitor Tablets, EDTA-free – Thermo Fisher #A32965 (1 tablet per 50 mL lysis buffer)
  • THAL-SNS-032 – Tocris #6532
  • Anti-CDK9 antibody – Cell Signaling Technology #2316
  • Anti-β-actin antibody – Bio-Techne #MAB8929
Instrumentation and Consumables
  • CyBio FeliX Basic Unit with Enclosure (Analytik Jena GmbH) (OL5015-24-100)
  • CyBio FeliX CHOICE Head (OL3316-14-250)
  • 8-channel CHOICE Adapter; 10-1000 µL (OL3316-14-330)
  • 8-channel CHOICE Adapter; 1-50 µL (OL3316-14-332)
  • 2 x Adapter 24 tubes, passive cooling function (844-00136-0)
  • CyBio Waste box (844-00430-0)
  • CyBio TipBox 96/250 µL Sterile (OL3811-25-637-S)
  • CyBio TipBox 96/50 µL Non-sterile (OL3811-25-535-N)
  • Adapter 96, passive cooling function (844-00135-0)
  • 2 x Height adapter 40 mm (844-00445-0)
  • 12-channel Reservoir Plate – Alphalabs #LW9400
  • Simple-Western Jess
  • Perkin-Elmer EnSpire – Reading plate at 562 nm
  • 1.5 mL Eppendorf Tubes – SLS #E0030123328
  • PCR tube strip and lids – Alphalabs #LW2535
  • 24-well plate – Thermo Fisher #930186
  • Polystyrene clear 384-well plate – Greiner #781101
  • Plate Seals – Alphalabs #LW2770
  • Bio-Rad MyCycler thermal cycler
  • Thermo Scientific Megafuge 8R + M10 microplate swinging bucket rotor
  • 37 °C incubator – Stuart Scientific incubator SI 18
Cell treatment and lysate preparation

MV-4-11 cells are seeded in a 24-well sterile polystyrene plate and incubated at 37 °C with 5% CO2 and humidity.
The cells are treated with serially diluted SNS-THAL-032 in technical duplicate before being collected in 1.5 mL tubes. The cells are washed in PBS before being lysed in 1x complete RIPA lysis buffer, incubated on ice for 30 minutes then clarified at 17,000 × g for 10 minutes at 4ºC. 

Bicinchoninic Acid (BCA) Assay (CyBio FeliX)

The BCA assay is a method of determining a cell lysate’s protein concentration by extrapolating unknown values from a standard curve constructed from samples with a known concentration of a specific protein (in this case BSA) in an appropriate buffer. For this assay BSA concentrations 
ranging from 2.0-0.025 mg/mL (Table 1) were used to determine the unknown protein concentrations from 24 unique samples at a time.