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STLCC_CPLS;Morrison 3/28/2011Page 1

Prepared by: Bob Morrison

STLCC_CPLS, Instrumentation SpecialistDec 08, rev Feb 09

BioRad IEF BioRad PowerPac HC

BioRad Criterion Cell

BioRad Protean II xiProteomics2D Gel Electrophoresis

Protocol

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STLCC_CPLS;Morrison 3/28/2011Page 2

Proteomics : BioRad 2-DSystems Overview

Link to BioRad IEF Methodsand Products Manual (pdf)

BioRad PowerPac HC

http://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_Methods_Manual.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_Methods_Manual.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_Methods_Manual.pdf

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STLCC_CPLS;Morrison 3/28/2011Page 3

Proteomics : BioRad Protean IEF Cell, Basic Features

The PROTEAN IEF cell is a dedicated instrument for running IPG strips.

High-capacity running platform accommodates up to twenty-four 7 cm strips or twelve 11, 17, 18, or 24

cm strips to increase eff iciency and throughput

Precision temperature control with a Peltier-cooled platform ensures reproducible focusing and an

expanded operating range of 1025C

Integrated 10,000 V, 2.4 mA power supply helps achieve the desired volt-hours faster when maximum

voltage is reached

Disposable rehydration/equilibration trays simplify the transfer of rehydrated strips into the focusing tray,

and have the added benefit of ensuring that residual sample buffer does not interfere with the current

path through the strips and adversely affect results

Tray options simplify handling. Each tray holds 112 IPG strips in individual numbered channels. For a

fully automated approach, rehydrate and run in the focusing tray. To increase throughput, rehydrate

strips in a rehydration/equilibration tray while running another set of IPG strips in the focusing tray in the

cell

Preset methods and real-time editing give maximum flexibility. The text-based user interface allows easy

programming of methods and provides a continuous update of run progress

Optional thermal printer records critical run data and provides a permanent record of the programmed

method and all run events. Run data are collected via the RS-232 serial port

Link to BioRad IEF Cell Instruction Manual (pdf)

http://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_BioRad_IEF_Cell.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_BioRad_IEF_Cell.pdf

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STLCC_CPLS;Morrison 3/28/2011Page 4

Proteomics : BioRad PowerPac HC Basic Features

PowerPac HC power supply, with 3.0 A of current and 300 W of power. In

addition to running standard horizontal agarose and vertical polyacrylamide

gels, the PowerPac HC power supply easily handles all your blotting

applications. To help you optimize every run, the PowerPac HC power

supply provides fully adjustable voltage, current, and power controls.

Runs 4 cells simultaneously

Timer-controlled

Fully programmable

CE and EN-61010 safety compliant

LCD screen shows all parameters during run

Case is stackable

Link to BioRad PowerPac Instruction Manual (pdf)

http://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_Powerpac.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_Powerpac.pdfhttp://www.bio-rad.com/cmc_upload/0/000/038/806/4110009A.pdf

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Proteomics: ReadyPrep 2-D Starter Kit Instructions

STLCC_CPLS;Morrison 3/28/2011Page 5

Link to BioRad

ReadyPrep 2-DInstruction Manual (pdf)

http://www.bio-rad.com/cmc_upload/0/000/038/806/4110009A.pdfhttp://www.bio-rad.com/cmc_upload/0/000/038/806/4110009A.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_ReadyPrep2D_Manual.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_ReadyPrep2D_Manual.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_ReadyPrep2D_Manual.pdfhttp://localhost/var/www/apps/conversion/current/tmp/scratch9770//dragon/users_wwwroot/Departments/fvbio/Proteomics_ReadyPrep2D_Manual.pdfhttp://www.bio-rad.com/cmc_upload/0/000/038/806/4110009A.pdf

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Proteomics: Protean II xiRemove Comb & Clamps

STLCC_CPLS;Morrison 3/28/2011Page 6

Remove Comb carefully from

one end

Remove sandwich clamp fromend while holding plate

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Proteomics: ProteanII xiRemove Clamps and Rinse

STLCC_CPLS;Morrison 3/28/2011Page 7

Remove other sandwich clamp

Thoroughly rinse edge of wellswith DI water.

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Proteomics: ProteanII xiRemount clamps

STLCC_CPLS;Morrison 3/28/2011Page 8

Remount plates in sandwichclamps and lower assemblyinto upper slots first

While pressing down andforward lock sandwich clamps

into fixture

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Proteomics: ProteanII xiLoad plates to Cell

STLCC_CPLS;Morrison 3/28/2011Page 9

Install other plate on sandwichclamps, ensure that theindented edge will be facing intoward the fixture center.

Load both plates withsandwich clamps into cell

center fixture.

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Proteomics: Protean II xiFill outer wells with Buffer

STLCC_CPLS;Morrison 3/28/2011Page 10

Load the entire sandwich clampassembly into the cell

Fill outer wells with buffer to alevel of about one half full

(must cover lower plate edge)

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Proteomics: Protean II xiFill inner well with Buffer

STLCC_CPLS;Morrison 3/28/2011Page 11

Fill the inner well (between the

plates) completely full of buffer

Approximate Fill level for bufferin outer wells

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Proteomics: Mini-Protean Tetra CellUnlock Plates and remove

STLCC_CPLS;Morrison 3/28/2011Page 12

Cell with plates and comb

Open doors to forward

position to release plates

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Proteomics: Mini Protean Tetra Cell Remove comb

STLCC_CPLS;Morrison 3/28/2011Page 13

Remove the comb carefully

from one end

Approximate Fill level for bufferin outer wells

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Proteomics: Mini Protean Tetra cellLoad Front side plate

STLCC_CPLS;Morrison 3/28/2011Page 14

In-step side of plate withchannels goes toward inner

Wet seal line with buffer andinsert front plate.

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Proteomics: Mini Protean Tetra cellComplete Plate assembly

STLCC_CPLS;Morrison 3/28/2011Page 15

Hold plates in position beforeclosing door clamps

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Proteomics: Mini Protean Tetra cellLoad Plate Assembly in Cell

STLCC_CPLS;Morrison 3/28/2011Page 16

Close door end clamps

Add plate assembly to cellfixture

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Proteomics: Mini Protean Tetra CellFill wells with Buffer

STLCC_CPLS;Morrison 3/28/2011Page 17

Fill outer portion of cell with

buffer to about 1/3rd

level

Completely fill inner well areawith buffer

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STLCC_CPLS;Morrison 3/28/2011Page 18

Link to BioRad2-D In a DayProtocol (pdf)

Proteomics : BioRad 2D in a Day, Step 1

Proteomics : BioRad 2D in a Day Steps 2 3 4

http://www.bio-rad.com/2dinadayhttp://www.bio-rad.com/2dinadayhttp://www.bio-rad.com/2dinadayhttp://www.bio-rad.com/2dinaday

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STLCC_CPLS;Morrison 3/28/2011Page 19

Link to BioRad2-D In a DayProtocol (pdf)

Proteomics : BioRad 2D in a Day, Steps 2,3,4

http://www.bio-rad.com/2dinadayhttp://www.bio-rad.com/2dinadayhttp://www.bio-rad.com/2dinadayhttp://www.bio-rad.com/2dinaday

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Proteomics: ReadyPrep 2-D Flowchart

STLCC_CPLS;Morrison 3/28/2011Page 20

Proteomics: ReadyPrep 2 D Sample Prep Rehydration

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Proteomics: ReadyPrep 2-D, Sample Prep, Rehydration

STLCC_CPLS;Morrison 3/28/2011Page 21

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Proteomics: ReadyPrep 2-D, Load Trays, Remove IPG Cover Strip

STLCC_CPLS;Morrison 3/28/2011Page 22

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Proteomics: ReadyPrep 2-D, IPG Strips onto Samples

STLCC_CPLS;Morrison 3/28/2011Page 23

Proteomics: ReadyPrep 2-D Isoelectric Focusing

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Proteomics: ReadyPrep 2-D, Isoelectric Focusing

STLCC_CPLS;Morrison 3/28/2011Page 24

Proteomics: ReadyPrep 2-D Wetting Drain Focus Tray

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Proteomics: ReadyPrep 2-D, Wetting,Drain, Focus Tray

STLCC_CPLS;Morrison 3/28/2011Page 25

Proteomics: ReadyPrep 2-D Focus Tray Program

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Proteomics: ReadyPrep 2-D, Focus Tray Program

STLCC_CPLS;Morrison 3/28/2011Page 26

Proteomics: ReadyPrep 2-D 4 4 Completing IEF Staining

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Proteomics: ReadyPrep 2 D, 4.4 Completing IEF, Staining

STLCC_CPLS;Morrison 3/28/2011Page 27

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STLCC_CPLS;Morrison 3/28/2011Page 28

Proteomics: Trans-Blot Cell Assembly

S

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STLCC_CPLS;Morrison 3/28/2011Page 29

Proteomics: Trans-Blot- Prepare for Blotting, Steps 1-3

P i T Bl P i S 4 6

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STLCC_CPLS;Morrison 3/28/2011Page 30

Proteomics: Trans-Blot , Preparation, Steps 4-6

P t i T Bl t P ti St 7 9

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STLCC_CPLS;Morrison 3/28/2011Page 31

Proteomics: Trans-Blot , Preparation, Steps 7-9

Proteomics: ReadyPrep2D, E.coli after stain, Prep for SDS-PAGE

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Proteomics: ReadyPrep2D, E.coli after stain, Prep for SDS PAGE

STLCC_CPLS;Morrison 3/28/2011Page 32

P t i R d P 2 D SDS PAGE B ff I d II IPG

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Proteomics: ReadyPrep 2-D, SDS-PAGE Buffer I and II, IPG

STLCC_CPLS;Morrison 3/28/2011Page 33

Proteomics: ReadyPrep2D, SDS-PAGE

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y p ,

STLCC_CPLS;Morrison 3/28/2011Page 34

Proteomics ReadyPrep2D IPG Strip to SDS Page GEL

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Proteomics: ReadyPrep2D, IPG Strip to SDS-Page GEL

STLCC_CPLS;Morrison 3/28/2011Page 35

Proteomics: ReadyPrep2D, Into IPG well with Melted Agarose

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y p , g

STLCC_CPLS;Morrison 3/28/2011Page 36

Proteomics: ReadyPrep2D, 4.9 Staining

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y p , g

STLCC_CPLS;Morrison 3/28/2011Page 37

Proteomics: ReadyPrep2D, Imaging on Densiometer

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y p g g