Day 2 Morning

Comparative/Functional ProteomicsComparative/Functional Proteomics

General Workflow for MS General Workflow for MS Protein IdentificationProtein Identification

Sample Preparation

1st Dimension Focusing

2nd Dimension SDS PAGE

Mulichannel Imaging

In-gel Digestion

Mass Spectrometry

Database Searching

Analysis and Excision

Sample Quant/Clean-up

Sample Labeling

DIGE

Sample Preparation

1st Dimension Focusing

2nd Dimension SDS PAGE

Imaging

In-gel Digestion

Mass Spectrometry

Database Searching

Analysis and Excision

Sample Quant/Clean-up

Non- DIGE

CyDye labeling; Prep GelCyDye labeling; Prep Gel

“Dope” withUnlabeled Protein

Sample Application for IPG StripsSample Application for IPG Strips

IPG strips are cast and dehydrated for storage.

Rehydrated in the presence of sample or buffer forintroduction of sample during focusing.

Strip Rehydration Manifold Cup-loading

SDS-Sodium Dodecyl Sulfate

Stacking and Separation in a Discontinuous Gel

PREPARE A ‘CLUB SANDWICH’ GEL MOLDPREPARE A ‘CLUB SANDWICH’ GEL MOLD

Glass plates, spacers and clamps

Insert a Numbered Tab

PREPARE A ‘CLUB SANDWICH’ GEL MOLDPREPARE A ‘CLUB SANDWICH’ GEL MOLD

Parafilm layer to help seal

Measure 1.5 cm down from notch

PREPARE A ‘CLUB SANDWICH’ GEL MOLDPREPARE A ‘CLUB SANDWICH’ GEL MOLD

Pipet in the acrylamide

Overlay with butanol

SECOND DIMENSION (SDS PAGE)SECOND DIMENSION (SDS PAGE)

Equilibrate stripreducing bufferalkylating buffer

Lay strip onto SDS gelfill space with bufferdrop strip into positionpour off excess buffer

Lay on MW marker tabs

Seal with LMT agarose

Assemble and run

LAY IPG DRYSTRIP ONTO SDS SLAB GELLAY IPG DRYSTRIP ONTO SDS SLAB GEL

Lay on trimmed DryStrip

Remove excess buffer

LAY MW TAB ONTO SDS SLAB GELLAY MW TAB ONTO SDS SLAB GEL

Select a MW marker tab

Place tab onto gel

ANCHOR MW TAB ONTO SDS SLAB GELANCHOR MW TAB ONTO SDS SLAB GEL

LMT Agarose

Melt the LMT agarose

Pipet agarose onto strip

ASSEMBLE SDS SLAB GELASSEMBLE SDS SLAB GEL

Assemble the tank

Add running buffer

RUN SDS SLAB GELRUN SDS SLAB GELStirrer on; check for leaks

Power ‘on’

Processing Strips after Focusing; ALL PROCEDURES IN SEMI-DARKNESS

Prepare reducing and alkylating buffers (2mL/strip) Reducing buffer: dissolve 10mg DTT/1mL into stock SDS equilibration buffer Alkylating buffer: dissolve 25mg iodoacetamide/1mL SDS equilibration buffer

Pipet 2mL reducing buffer into a trough on the Rehydration Tray. One trough per strip.

Stop IEF; record total volthours: ~25,000 Vhr (13 cm) For all strips: remove strip from “coffin” w/ forceps, gently lay on Kimwipe

to remove excess cover fluid, place into trough with acrylamide side ‘up’. Shake tubes horizontally on platform for 15 min (make certain strips are

covered with buffer and rocking). Carefully aspirate reducing buffer. Pipet 2mL alkylating buffer into each trough. Shake as above.