Day 2 Morning. Comparative/Functional Proteomics.
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Transcript of Day 2 Morning. Comparative/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
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
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
ANCHOR MW TAB ONTO SDS SLAB GELANCHOR MW TAB ONTO SDS SLAB GEL
LMT Agarose
Melt the LMT agarose
Pipet agarose onto strip
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.