Dimensional Gel Operations

 
Brief Description of Materials and Equipment First Dimension (IEF)

Sample is desalted using Bio-Spin chromatography (gel filtration). The Bio-Spin columns 1 x 10 cm (Bio-Rad Laboratories, Hercules, CA) are packed in-house with Bio-Gel P series (Bio-Rad). Protein concentration is determined using a Coomassie® Plus Bradfort Assay reagent kit (Pierce, Rockford, IL). For diluted samples, the desalted solution is concentrated by either lyophilization or vacuum centrifugation. The concentrated sample is solubilized in rehydration buffer (7 M Urea, 2 M thiourea, 2% CHAPS, 0.8% ampholyte, 0.02% bromophenol blue, and 65 mM diththiothreitol [DTT]), loaded onto an appropriate immobilized pH gradient (IPG) and focused with Protein IEF cell (Bio-Rad).

   
Second Dimension (SDS-PAGE) 

Both large size format SDS-PAGE gels (takes 7, 11 or 17  cm IPG strips) and mini gels (for 7 cm IPG strips) are casted in-house. The small criterion gels (for 11 cm IPG strips) are purchased pre-cast (Bio-Rad). The IPG strips are equilibrated for 10 min with 2% (w/v) DTT in 4.2 ml/strip of an equilibration solution [0.38 M Tris-base, pH 8.8, 6 M urea, 2% (w/v) SDS and 20% (v/v) glycerol] and for 10 min with 2.5% (w/v) iodoacetamide in the equilibration solution. Each IPG strip is loaded onto the appropriate percentage acrylamide gel, sealed with 1% agarose in buffer (see below); and electrophoresed (100 or 200 V for 1 – 8 h, depending on the size of gel) using either mini gel apparatus (Biorad), Protean Plus or Criterion Dodeca cells (Bio-Rad) in a buffer that contains 25 mM Tris, 190 mM glycine and 0.1% SDS. After electrophoresis, proteins are fixed and are visualized using either Coomassie, Silver or Sypro Ruby stains.

   
Image Analysis 

Stained gels are scanned with the image system VERSADOC (Bio-Rad) using PDQuest software version 8.1 (Bio-Rad) and the proteins of interest are marked for excision. We currently have a site license for up to three users so that the requesting laboratory can analyze their own gels.