Simple Western Protocol

For use with Jess, Abby, Leo Systems, and similar Simple Western Systems

Simple Western assays are capillary-based immunoassays that automate the traditional western blot workflow, eliminating manual steps such as gel casting, transfer, and film exposure. Simple Western technology delivers high sensitivity, reproducibility, and quantitation with only 3 µL of input sample, producing fully analyzed results in as little as three hr. This protocol outlines key steps for performing protein analysis using Simple Western systems, including Jess, Abby, and Leo Systems. Cell Signaling Technology (CST) has rigorously validated a collection of antibodies for use in Simple Western assays. When using CST^® antibodies, it is crucial to consult the specific CST product webpage or datasheet for recommended dilution ranges, which were validated with the chemiluminescence detection mode.

A. Solutions and Reagents

Except for the user-supplied primary antibody, all necessary reagents—from sample preparation to signal detection—are included in the Simple Western Separation and Detection Modules.

1. Choose a Separation Module compatible with your instrument, detection mode (chemiluminescence or fluorescence), and the molecular weight range of your target.

   1. Chemiluminescence Separation Modules

      For Jess and Abby: 12-230 kDa 66-440 kDa 2-40 kDa

      For Leo: 12-230 kDa 66-440 kDa 2-40 kDa

   2. Fluorescence Separation Modules

      For Jess:

      • 2-40 kDa
      • 12-230 kDa
      • 66-440 kDa

2. Choose a Detection Module compatible with your primary antibody and detection mode.

   NOTE:

   • Chemiluminescence Detection Modules are available in formats supporting 200 or 600 capillaries and are compatible with Jess, Abby, and Leo.
   • Fluorescence Detection Modules are available in 200-capillary format and are only compatible with Jess.
   • For Leo, use 600-capillary Detection Modules for optimal throughput.

   1. Chemiluminescence Detection Modules

      For 200 capillaries: Anti-Rabbit Anti-Mouse Anti-Goat Anti-Human IgG No Secondary Biotin

      For 600 capillaries: Anti-Rabbit Anti-Mouse Anti-Goat Anti-Human IgG No Secondary Biotin

   2. Fluorescence Detection Modules

      Anti-Rabbit IR Anti-Mouse IR

      Anti-Rabbit NIR Anti-Mouse NIR

B. Sample Preparation

1. Prepare 400 mM DTT (clear tube). Pierce foil with a pipette tip, add 40 µL DI water, and mix gently.
2. Prepare 5X Master Mix (pink tube). Add 20 µL of 10X Sample Buffer and 20 µL of prepared DTT. Mix gently.
3. Lyse cells using RIPA buffer or other suitable lysis buffer with protease and phosphatase inhibitors. The optimal protein concentration depends on the expression level of your protein. Dilute lysates as necessary with 0.1X Sample Buffer.
4. Mix four parts lysate and one part 5X Master Mix. Gently mix by pipette and close the tube.
5. Vortex to mix. Heat at 95°C for 5 min. Vortex and spin samples.
6. Store prepared samples on ice.

C. Antibody Preparation

1. Dilute your primary antibody in Antibody Diluent 2 provided with the Detection Module. If using a goat primary antibody, dilute with Milk-Free Antibody Diluent provided.

NOTE: For CST primary antibodies, always refer to the product webpage or datasheet on the CST website (cellsignal.com) for recommended dilution ranges, which were validated with the chemiluminescence detection mode.

2. Secondary antibody is provided ready-to-use in the Detection Module.
3. Store prepared antibodies on ice.
4. Prepare Luminol-S and Peroxide (if applicable). Combine 200 μL Luminol-S and 200 μL Peroxide in a microcentrifuge tube, mix by pipette, and store on ice.

D. Plate Setup

1. Load the sample plate by referring to the product inserts provided for assay-specific plate preparation.

NOTE: Plate loading is compatible with various liquid handling automation solutions.

2. Centrifuge the sample plate for 5 min at 1,000 x g (~2,500 rpm), ensuring the liquid is fully down in the wells. For Leo runs, centrifuge the pre-filled reagent plates.

E. Instrument Run

1. Open Compass for Simple Western software, the control and data analysis application for Simple Western instruments.
2. Click New Assay and select the assay type and size range for your run, or choose Open Assay to select from a menu of saved assays.
3. Assign sample plate rows in the Layout pane and assay parameters in the Protocol pane. For Leo runs, click Assay Setup in the Sample Layout to specify required cartridges and sample plate rows.
4. Remove evaporation seals from the sample plate (Jess and Abby) or the pre-filled reagent plate (Leo).
5. Load the prepared sample plate and capillary cartridge into the instrument. For Leo runs, load the pre-filled reagent(s) plate in the pre-filled reagent plate holder(s). Close the instrument door.
6. Click the Start button in Compass. The instrument will automatically perform:
1. Capillary electrophoresis
2. Protein immobilization to the capillary wall
3. Blocking
4. Primary antibody incubation
5. Secondary antibody incubation
6. Signal detection and image/data capture
7. Discard capillary cartridges and plates when the run is complete.

F. Data Analysis

• Upon run completion, Compass for Simple Western automatically provides electropherograms, lane view images, and a quantitative results table of your data.
• Use built-in tools to annotate data, generate standard curves, and normalize protein expression.
• Refer to the Compass for Simple Western User Guide for more information.

To learn more about Simple Western technology and find related resources, visit: bio-techne.com/simplewestern.

For Research Use Only. Not for Use in Diagnostic Procedures.

Simple Western, Jess, Abby, Leo, and Compass for Simple Western are trademarks and/or registered trademarks of ProteinSimple.

Contact us with any questions