Proteomics 2D Gel Electrophoresis - STLCC.eduusers.stlcc.edu/...2DGel_Electrophoresis.pdf · Dec...

STLCC_CPLS;Morrison 3/28/2011

Prepared by: Bob Morrison

STLCC_CPLS, Instrumentation Specialist

Dec 08, rev Feb 09

BioRad IEF BioRad PowerPac HC

BioRad Criterion Cell

BioRad Protean II xi Proteomics

2D Gel Electrophoresis

Protocol


Proteomics : BioRad 2-D

Systems Overview

Link to BioRad IEF Methods

and Products Manual (pdf)

BioRad PowerPac HC

//dragon/users_wwwroot/Departments/fvbio/Proteomics_Methods_Manual.pdf


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 efficiency and throughput

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

expanded operating range of 10–25°C

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 1–12 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)

//dragon/users_wwwroot/Departments/fvbio/Proteomics_BioRad_IEF_Cell.pdf


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)

//dragon/users_wwwroot/Departments/fvbio/Proteomics_Powerpac.pdf

Proteomics: ReadyPrep 2-D Starter Kit Instructions


Link to BioRad

ReadyPrep 2-D

Instruction Manual (pdf)

http://www.bio-rad.com/cmc_upload/0/000/038/806/4110009A.pdf

//dragon/users_wwwroot/Departments/fvbio/Proteomics_ReadyPrep2D_Manual.pdf

Proteomics: Protean II xi

Remove Comb & Clamps


Remove Comb carefully from

one end

Remove sandwich clamp from

end while holding plate

Proteomics: ProteanII xi

Remove Clamps and Rinse


Remove other sandwich clamp

Thoroughly rinse edge of wells

with DI water.


Remount clamps


Remount plates in sandwich

clamps and lower assembly

into upper slots first

While pressing down and

forward lock sandwich clamps

into fixture


Load plates to Cell


Install other plate on sandwich

clamps, ensure that the

indented edge will be facing in

toward the fixture center.

Load both plates with

sandwich clamps into cell

center fixture.


Fill outer wells with Buffer


Load the entire sandwich clamp

assembly into the cell

Fill outer wells with buffer to a

level of about one half full

(must cover lower plate edge)


Fill inner well with Buffer


Fill the inner well (between the

plates) completely full of buffer

Approximate Fill level for buffer

in outer wells

Proteomics: Mini-Protean Tetra Cell

Unlock Plates and remove


Cell with plates and comb

Open “doors” to forward

position to release plates

Proteomics: Mini Protean Tetra Cell

– Remove comb


Remove the comb carefully

from one end

Approximate Fill level for buffer

in outer wells

Proteomics: Mini Protean Tetra cell

Load Front side plate


In-step side of plate with

channels goes toward inner

Wet seal line with buffer and

insert front plate.


Complete Plate assembly


Hold plates in position before

closing door clamps


Load Plate Assembly in Cell


Close door end clamps

Add plate assembly to cell

fixture

Proteomics: Mini Protean Tetra Cell

Fill wells with Buffer


Fill outer portion of cell with

buffer to about 1/3rd level

Completely fill inner well area

with buffer


Link to BioRad

2-D In a Day

Protocol (pdf)

Proteomics : BioRad 2D in a Day, Step 1

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


Link to BioRad

2-D In a Day

Protocol (pdf)

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

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

Proteomics: ReadyPrep 2-D Flowchart


Proteomics: ReadyPrep 2-D, Sample Prep, Rehydration


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


Proteomics: ReadyPrep 2-D, IPG Strips onto Samples


Proteomics: ReadyPrep 2-D, Isoelectric Focusing


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


Proteomics: ReadyPrep 2-D, Focus Tray Program


Proteomics: ReadyPrep 2-D, 4.4 Completing IEF, Staining



Proteomics: Trans-Blot Cell Assembly


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


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


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

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


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


Proteomics: ReadyPrep2D, SDS-PAGE


Proteomics: ReadyPrep2D, IPG Strip to SDS-Page GEL


Proteomics: ReadyPrep2D, Into IPG well with Melted Agarose


Proteomics: ReadyPrep2D, 4.9 Staining


Proteomics: ReadyPrep2D, Imaging on Densiometer


Proteomics 2D Gel Electrophoresis - STLCC.eduusers.stlcc.edu/...2DGel_Electrophoresis.pdf · Dec...

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