Institutional Development Award
|Contact Information for Nevada Proteomics Center|
|Phone||(775) 784-6337: 2-D Gels|
|Phone||(775) 784-1590: Mass Spectrometry|
|Location||School of Medicine
Manville Medical Building, Rooms 15-17, MS 0330
|Address||1664 N. Virginia Street
Reno, NV 89557-MS 0330
Below is an example of Materials and Methods that could be included in publications that describe use for either standard 2-D gels or DIGE gels. Some of the details of gels or running conditions will vary with individual experiments. Please contact us for refinement of Materials and Methods before submitting in a publication.
All reagents and solutions for 2-D were obtained from Bio-Rad (Hercules, CA) except as noted.
200 ul of each extract was loaded onto a 3-10 11 cm IPG strip by overnight passive rehydration. Isoelectric focusing was carried out on a Bio-Rad Protean i12 cell using a program as follows: 250 V, linear ramp for 20 minutes; 8000 V, linear ramp for 2 hours 30 minutes; and 8000 V for a total of 20,000 Vhr (all steps with a maximum current of 50 uA per gel). Strips were stored at –80°C, then thawed and incubated twice for 10 minutes each in 8M urea, 2% SDS, 0.05 M TrisHCl, pH 8.8, 20% glycerol. The first incubation contained 2% DTT and the second contained 2.5% iodoacetamide. The strips were then layered on 8-16% Criterion Tris HCl gradient gels and embedded in place with 0.5% agarose, along with Precision Plus Protein Unstained Standards. Electrophoresis was performed at a constant current of 200 mA until the dye front ran off the gel. Gels stained overnight with either Bio-Safe Coomassie or Sypro Ruby stain.
Stained gels were imaged on a Bio-Rad ChemiDoc MP imager. Images of gels were compared using Bio-Rad PDQuest version 8.0.1 software and spot sets were created. The spots in these sets were excised from gels using a Bio-Rad EXQuest Spot Cutter.
IPG strips and electrophoresis chemicals were obtained from Bio-Rad (Hercules, CA). Electrophoresis gels were purchased from Jule (Milford, CT). The DeStreak Reagent and CyDyes are products of GE Healthcare. The EZQ Protein Assay was obtained from Invitrogen.
Pellets of precipitated protein were dissolved in 200 μl DIGE Reaction Buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM TrisHCl, pH 8.7) and assayed for protein concentration by the EZQ Protein Assay (Invitrogen). Each sample was diluted to between 1.0 and1.5 μg protein/μl using DIGE Reaction Buffer. 60 μg of each sample was transferred to a 0.5 ml microfuge tube and placed on ice. A standard pool of all samples was prepared by combining 30 μg of each sample in a microfuge tube. The three CyDyes were dissolved in dimethylformamide to give a final concentration of 0.2 nmole/μl. To each of the protein samples was added 1.34 μl 0.2 nmole/μl of either Cy3 or Cy5. To the standard pool was added 1.34 μl Cy2 per 60 μg protein. The samples were placed back on ice and reacted in the dark for 30 minutes. The reaction was terminated by the addition of 2 μl 10 mM lysine, followed by an additional 10 minutes incubation in the dark.
For each gel to be run, 53 μg of one Cy3 labeled sample was combined with 53 μg of one Cy5 labeled sample and 53 μg of the Cy2 labeled standard was added. To the mixture is added 2.4 μl 3-10 ampholytes, 4 μl 0.1% Bromophenol Blue and 5.76 μl DeStreak Reagent (GE Healthcare). The final volume was adjusted to 480 μl with DIGE Dilution Buffer (7 M urea, 2 M thiourea, 4% CHAPS). 450 μl of the mixture was used to rehydrate a 24 cm pH 3-10 IPG strip by passive overnight rehydration. The strips were then focused at a maximum voltage of 7500 V for 85,000-100,000 Vhrs. The strips were equilibrated twice, for 12 minutes each, in Equilibration Base Buffer (6 M urea, 2% SDS, 0.06 M Tris HCl, pH 8.8, 20% glycerol). The first equilibration step contained 2% DTT and the second contained 2.5% iodoacetamide. The strips were run on 20 x 26 cm 12% polyacrylamide Tris-HCl gels. The gels were run at 40 V for 2 hours and then 100 V until the dye front reached the bottom of the gel.
Gels were imaged with a Typhoon Trio imager (GE Healthcare) using the suggested settings for the three CyDyes. Analysis was performed with DeCyder v7 software (GE Healthcare) and spots are picked with a Digilab ProPic II spot cutter with DIGE.
Updated October 2013