let’s get started!bosch.test.med.usyd.edu.au/facilities/molecular-biology/... · 2017-08-25 ·...

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Reagents and materials for each module

ORANGE BOX — STORE AT 18-24 °C

INCLUDESKIT COMPONENT PART NUMBER

Wash Buffer (60 mL) 042-202

10X Sample Buffer (440 μL) 042-195

Pre-Filled Microplates with Split Running Buffer (8 or 2 each)

043-165

Capillary Cartridges (8 or 2 each) 009-050

CLAMSHELL — STORE AT 2-8 °C

INCLUDESKIT COMPONENT PART NUMBER

Standard Pack: BiotinylatedLadder, Fluorescent 5X Master Mix, DTT, and empty 0.6 mL tube (8 or 2 each)

PS-ST01

Other things you’ll need• A Detection Module for Wes, Peggy Sue or Sally Sue• Protein samples • Primary antibodies• Water, 0.22 µm-filtered and deionized (molecular biology

grade or better)• Pipettes and tips• Microcentrifuge and tubes• Ice and ice bucket• Vortex• Heat block• Centrifuge with plate adapter

A few things you should know• Warm Wes’ plates up to room temperature for at least 24

hours before you start the first assay.

• Capillaries are moisture- and light-sensitive.

• Store unopened cartridge packages and plates at room temperature and do not remove the seals until ready to use.

• The first capillary in the cartridge has been optimized for running the biotinylated ladder. Pipette the biotinylated ladder and samples only as shown in Step 2.

• Plate well evaporation dramatically affects experimental results. To prevent evaporation, keep the lid on the assay plate and do not remove the evaporation seal until you’re ready to put the assay plate into Wes. Keep the lid on between reagent additions and post-preparation.

• An optional System Control Primary Antibody (042-196 or 042-191) can be mixed with your primary antibody in the assay to calculate inter-assay and inter-instrument variability.

• You can use Bicine/CHAPS buffer (P/N: 040-764) or RIPA buffer (P/N: 040-483) to lyse your cells.

Let’s get started!

12-230 kDa Wes Separation ModuleModule with 2 x 25 capillary cartridges (Part# SM-W003)Module with 8 x 25 capillary cartridges (Part# SM-W004)

B PREPARE YOUR SAMPLES

• The optimal protein concentration depends on the expression level of your protein. Refer to training material for concentration recommendations. If needed, dilute your lysate with 0.1X Sample Buffer (dilute 10X Sample Buffer 1:100 with water).

• Combine 1 part (e.g. 1 μL) 5X Fluorescent Master Mix with 4 parts (e.g. 4 μL) lysate in a microcentrifuge tube (final concentration 0.2 mg/mL).

• Pierce foil withpipette tip

• Gently mixby pipette





• 1 part 5XFluorescentMaster Mix

• 4 parts lysate


• Close tube

C DENATURE YOUR SAMPLES AND BIOTINYLATED LADDER

Transfer entirevolume to0.6 mL tube

Gently pipetto resuspend

Open, remove4 tubes

Gently pipetteup and downto mix

Store on ice

Vortex to mix

Vortex to mix

Store on ice

Ladder

Ladder 5X Fluor.Master

DTT

Sample

• Vortex to mix

• 95° C, 5 minutes

• Vortex

• Spin

Close tube

0.6 mL

Standard Pack

D PREPARE REAGENTS FROM DETECTION MODULE

Substrate

• Combine 200 µL Luminol-S and 200 µL Peroxide supplied in the detection module in a microcentrifuge tube.



Open, remove4 tubes


Store on ice

Vortex to mix

Vortex to mix

Store on ice

Ladder


DTT

Sample

• Vortex to mix


• Vortex

• Spin

Close tube

0.6 mL

Standard Pack

A PREPARE STANDARD PACK REAGENTS



Open, remove4 tubes


Store on ice

Vortex to mix

Vortex to mix

Store on ice

Ladder


DTT

Sample

• Vortex to mix


• Vortex

• Spin

Close tube

0.6 mL

Standard Pack

DTT (Clear Tube)








• 4 parts lysate


• Close tube

• Add 40 µL deionized water to make a 400 mM solution

Fluorescent 5X Master Mix (Pink Tube)








• 4 parts lysate


• Close tube

• Add 20 µL 10X Sample buffer • Add 20 µL prepared 400 mM DTT solution

Biotinylated Ladder (White Tube)








• 4 parts lysate


• Close tube

• Add 16 µL deionized water• Add 2 µL 10X Sample Buffer• Add 2 µL prepared 400 mM DTT solution



Open, remove4 tubes


Store on ice

Vortex to mix

Vortex to mix

Store on ice

Ladder


DTT

Sample

• Vortex to mix


• Vortex

• Spin

Close tube

0.6 mL

Standard Pack

1. Prepare your reagents

Antibodies (skip if running Total Protein Assay)

• Primary Antibody: Dilute in Antibody Diluent 2. Refer to training material to determine optimal dilution. If you’re using the System Control, add 25 μL of the System Control Primary Antibody to 225 μL of your diluted Primary Antibody. Pipette 10 μL of the mixture into each Primary Antibody well.

• Secondary HRP Conjugate: It is provided ready to use in the Detection Module. If you are using your own, consult your FAS or the Antibody Database on the ProteinSimple website.

1. Load the desired assay in Compass software v3.1 or higher.

2. Open Wes’ door.3. Insert a capillary cartridge into the cartridge holder.

The interior light will change from orange to blue.4. Remove the assay plate lid. Hold plate firmly on

bench and carefully peel off evaporation seal. Pop any bubbles observed in the Separation Matrix wells with a pipette tip.

5. Place the assay plate on the plate holder.6. Close Wes’ door.7. Click the Start button in Compass.8. When the run is complete, discard the plate and

cartridge.

• For more consistent results, keep the lid on between reagent additions and minimize bubble formation when adding Wash Buffer to the troughs in the microplates.

1. Dispense reagents into the assay plate using the volumes shown in the plate diagram.

2. Centrifuge the plate for 5 minutes at 2500 rpm (~1000 x g) at room temperature. Ensure liquid is fully down in all wells.

Cartridge holder

Plate holder

2. Pipette your plate (Immunoassay)

3. Start Wes

1 5 10 15 20 25A

B

C

D

E

F

Evaporation sensitive

Peel off immediately before placing in instrument

Biotinylated Ladder, 5 μL; Prepared Samples, 3 μL

Wes Antibody Diluent 2, 10 μL

Wes Antibody Diluent 2, 10 μL; Primary Antibody, 10 μL

Streptavidin-HRP, 10 μL; Secondary HRP Conjugate, 10 μL

Luminol-Peroxide Mix, 15 μL

Wash Bu�er500 µL/compartment2.5 mL/row

let’s get started!bosch.test.med.usyd.edu.au/facilities/molecular-biology/... · 2017-08-25 ·...

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