Toll-free: (888) 607-9692
Tel: (408) 510-5500
Fax: (408) 510-5599
[email protected]
proteinsimple.com
© 2014 ProteinSimple. ProteinSimple, Simple Western,
Wes and the ProteinSimple logo are trademarks and/or
registered trademarks of ProteinSimple.
043-026 RevA
Total Protein Master Kit
12–230 kDa Total Protein Master Kit (Part# PS-TP01)
66–440 kDa Total Protein Master Kit (Part# PS-TP02)
Let’s get started!
Reagents and materials in this kit
Other things you’ll need
BOX 1 — STORE AT ROOM TEMPERATURE
• Protein samples
• 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
INCLUDES
PART NO
Wash Buffer (60 mL)
042-202
10X Sample Buffer (440 μL)
042-195
Pre-Filled Microplates (8)
• 12–230 kDa or
• 66–440 kDa
PS-PP01
PS-PP02
Capillary Cartridges (8)
PS-CC01
A few things you should know
• Bring the Total Protein Labeling Reagent and Reconstitution
Agents 1 and 2 to room temperature before use.
BOX 2 — STORE AT 4°C
INCLUDES
PART NO
Antibody Diluent 2 (20 mL)
042-203
Luminol-S (1.5 mL)
042-233
Peroxide (1.5 mL)
042-234
Total Protein Streptavidin-HRP
042-976
Standard Pack (8): Biotinylated
Ladder 1, Fluorescent 5X Master Mix,
DTT, and empty 0.6 mL tube
• Standard Pack 1 (12–230 kDa) or
• Standard Pack 3 (66–440 kDa)
PS-ST01
PS-ST03
Total Protein Reconstitution Agent 2
042-975*
BOX 3 — STORE AT –80°C
INCLUDES
PART NO
Biotin Labeling Reagent plus Total
Protein Reconstitution Agent 1
042-973*
*Use part # PS-BL01-8 to re-order these two
reagents a la carte
• 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 seal until you’re ready to put the assay plate
into Wes. Keep the lid on between reagent additions and postpreparation.
• Biotin Labeling Reagent is sensitive to moisture. Pierce foil only
when ready to use. Keep unused tubes in the sealed bag at
-70 °C.
• The Antibody Diluent 2, Sample Buffer (10X), Total Protein
Streptavidin-HRP, Wash Buffer, and reagents in the Total Protein
Biotin Labeling kit are ready-to-use reagents and should not be
diluted.
• You can use Bicine/CHAPS buffer (P/N: 040-764) or RIPA buffer
(P/N: 040-483) to lyse your cells.
1. Prepare your reagents
A
B
PREPARE STANDARD PACK REAGENTS
• Pierce foil with
pipette tip
Standard Pack
• Gently mix
by pipette
Open, remove
4 tubes
Ladder 5X Fluor.
Master
DTT
0.6 mL
Gently pipet
DTT (Clear
Tube)
to resuspend
• by
Pierce
foil with
pipette
pipette tip
• Gently mix5X
Fluorescent
by pipette
0.6 mL
• Pierce foil with
pipette tip
• Gently mix
by pipette
Ladder 5X Fluor.
Master
DTT
0.6 mL
Transferpipet
entire
Gently
PREPARE
YOUR SAMPLES
volume
to
to
resuspend
Close tube
0.6 mL tube
• When using lysates, we recommend a final lysate
concentration of 0.2 mg/mL. The optimal protein
• Pierce foil with
concentration
depends on the expression level of your
pipette tip
•
Gently
mix
protein. If needed, dilute your lysate with 0.1X Sample
by pipette
entire
BufferTransfer
(dilute
10X Sample Buffer 1:100 with water).
volume to
Close5X
tubeFluorescent Master Mix
0.6 mL1tube
• Combine
part (e.g. 1 μL)
to mix
with 4Vortex
parts
(e.g. 4 μL) lysate in a microcentrifuge tube
(final concentration 0.2 mg/mL).
C
• Gently mix
by pipette
• Close tube
Store on ice
Vortex to mix• Vortex to mix
• 95° C, 5 minutes
• Vortex
• Spin
Transfer entire
volume to
• Pierce
foil
• Add
40
µLwith
deionized water
to make a
Close tube
0.6 mL tube
pipette tip
400 mM
solution
• Gently mix
DTT
Open, remove
4 tubes
Gently pipet
to resuspend
Standard
Pack
• 1 part 5X
Fluorescent
Master Mix
• 4 parts lysate
• Pierce foil with
pipette tip
• Gently mix
by pipette
Ladder 5X Fluor.
Master
DENATURE YOUR SAMPLES AND
BIOTINYLATED LADDER
Ladder Sample
Master Mix (Pink Tube)
Store on ice
• Vortex to mix
• 95° C, 5 minutes
• Vortex
• Spin
• Pierce foil with
pipette tip
• Gently mix
by pipette
Vortex to mix
• Pierce foil with
pipette tip
• 1 part 5X • Gently mix
Fluorescent by pipette • Gently mix
Vortex to mix
Ladder Sample
• Add 20 µL 10X Sample buffer
by pipette
20 µL •prepared
400 mM DTT solution
Close tube
D
Master Mix
• Add
• 4 parts lysate
Open, remove
4 tubes
Store on ice
• Pierce foil with • Vortex to mix
Standard Pack
pipette tip Ladder
Biotinylated
(White
Tube)
• 95° C,
5 minutes
• Gently mix
• Vortex
• Pierce
foil with
by pipette
Ladder 5X Fluor. DTT
• Spin
pipette tip
Master
• Gently mix
by pipette
MIX LUMINOL-S AND PEROXIDE
• Combine 150 µL Luminol-S and 150 µL Peroxide in a
microcentrifuge tube
Vortex to mix
0.6 mL
Store on ice
Vortex to mix
Ladder Sample
Gently pipet
to resuspend
• Pierce
• Add
16foil
µLwith
deionized water
• Addpipette
2 µLtip10X Sample Buffer
• Gently mix
• Addby2pipette
µL prepared 400 mM DTT solution
• 1 part 5X
Fluorescent
Master Mix
• 4 parts lysate
• 1 part 5X
Fluorescent
Master Mix
• 4 parts lysate
• Gently mix
Vortex to by
mix
pipette
Transfer entire
•
Close
tube
volume to
0.6 mL tube
• Gently mix
by pipette
• Close tube
Vortex to mix
Vortex to mix
E
PREPARE YOUR LABELING REAGENT
Store on ice
• The Biotin
Labeling
Reagent should only be prepared
Vortex to
mix
immediately before loading the plate
Close tube
• Pierce foil with
pipette tip
• Add 150 µL
Reconstitution
Agent 1
(white cap)
• Gently mix
by pipette
• Add 150 µL
Reconstitution
Agent 2
(orange cap)
• Gently mix
by pipette
Store on ice
• Store the reagent at room temperature until ready to
be dispensed into the assay plate.
2. Pipette your plate
A
1
5
10
15
20
25
Biotinylated Ladder, 5 μL;
Prepared Samples, 5 μL
B
Wes Antibody Diluent 2, 10 μL;
Total Protein Labeling Reagent, 10 μL
C
Wes Antibody Diluent 2, 10 μL
D
Total Protein Streptavidin-HRP, 8 μL
E
Luminol-Peroxide Mix, 10 μL
F
Wash Buffer
500 µL/compartment
2.5 mL/row
Evaporation sensitive
Peel off immediately before
placing in instrument
• 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.
3. Start Wes
1. Load the Total Protein assay in Compass software.
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.
Cartridge
holder
Plate
holder