General Workflow for MS Protein Identification Sample Preparation 1 st Dimension Focusing 2 nd Dimension SDS PAGE Mulichannel Imaging In-gel Digestion Mass Spectrometry Database Searching Analysis and Excision Sample Quant/Clean-up Sample Labeling DIGE Sample Preparation 1 st Dimension Focusing 2 nd Dimension SDS PAGE Imaging In-gel Digestion Mass Spectrometry Database Searching Analysis and Excision Sample Quant/Clean-up Non- DIGE
CyDye labeling; Prep Gel “Dope” with Unlabeled Protein
Sample Application for IPG Strips IPG strips are cast and dehydrated for storage. Rehydrated in the presence of sample or buffer for introduction of sample during focusing. Strip Rehydration ManifoldCup-loading
PREPARE A ‘CLUB SANDWICH’ GEL MOLD Glass plates, spacers and clamps Insert a Numbered Tab
PREPARE A ‘CLUB SANDWICH’ GEL MOLD Parafilm layer to help seal Measure 1.5 cm down from notch
PREPARE A ‘CLUB SANDWICH’ GEL MOLD Pipet in the acrylamide Overlay with butanol
SECOND DIMENSION (SDS PAGE) Equilibrate strip reducing buffer alkylating buffer Lay strip onto SDS gel fill space with buffer drop strip into position pour off excess buffer Lay on MW marker tabs Seal with LMT agarose Assemble and run
LAY IPG DRYSTRIP ONTO SDS SLAB GEL Lay on trimmed DryStrip Remove excess buffer
LAY MW TAB ONTO SDS SLAB GEL Select a MW marker tab Place tab onto gel
ANCHOR MW TAB ONTO SDS SLAB GEL LMT Agarose Melt the LMT agarose Pipet agarose onto strip
ASSEMBLE SDS SLAB GEL Assemble the tank Add running buffer
RUN SDS SLAB GEL Stirrer 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.
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