Approximate timeline
Day 1
Immobilized pH gradient (IPG) strip rehydration: active time 5 min per sample, rehydration is carried out overnight
Day 2
Isoelectric focusing (IEF): active time 5 min per sample, IEF 5-6 hrs.
Preparation for the 2nd dimension SDS PAGE: 1 hr.; necessary reagents and solutions are prepared during the last hour of IEF.
SDS PAGE, staining, and de-staining: 2.5 hrs. with de-staining overnight.
Brief procedure (learn more in the 2D PAGE manual )
Day 1 (late afternoon)
Protein sample should be lyophilized or precipitated. To keep the ionic strength of the protein solution at minimum, avoid salts. Non-ionic and zwitterionic solubilizing agents could be present, but keep in mind solubility limits.
Protein load for a complex sample to be stained with Coomassie is approximately 100 ug. The protein load varies depending on many factors including stain sensitivity, IPG range, or downstream applications (refer to the manual).
Protein sample is dissolved in the sample buffer (Bio-Rad cat #163-2106), 125 ul for 7 cm IPG strips and 185 uL for 11 cm IPG strips. The protein solution is pipetted into a channel in a rehydration tray. An IPG strip is placed gel side down into the channel, covered with mineral oil, and left overnight at 4 C.
Day 2 (early morning)
IPG strips are placed into a focusing tray, covered with mineral oil, and the tray is placed into the IEF cell for 5-6 hrs.
After the IEF is complete, strips are soaked in a reducing buffer, followed by alkylating buffer, rinsed in SDS running buffer, and placed at the top of an SDS-PAGE gel. A molten agarose solution is applied to the well. Once agarose solidifies, the gel is ready for the 2nd dimension electrophoresis.
After completion of the SDS-PAGE, gels are rinsed, stained, and destained. Alternatively, the gels can be electro-blotted.
Supplies
What IPG strip is the best for your experiment? Start by choosing the length (7 cm or 11 cm). Short strips are compatible with the Mini-PROTEAN cell, longer strips are compatible with the Criterion cell. All parameters being equal, a larger gel affords better resolution. Next select pH range depending on a type of your experiment: broad range (e.g. 3-10) for a global view, narrow range for a zoom-in view.
Bio-Rad cat #163-2000, 7 cm, pH 3–10, immobilized pH gradient (IPG) strip for first-dimension separations, pkg of 12
Bio-Rad cat #163-2014, 11 cm, pH 3–10, immobilized pH gradient (IPG) strip for first-dimension separations, pkg of 12
Buffers and gels
Rehydration/sample buffer, Bio-Rad cat #163-2106
Agarose, Bio-Rad cat #163-2111
Criterion Tris-HCl Gel, Bio-Rad cat #345-0040, Pkg of 1, 8–16% polyacrylamide gel, prep+2 well, 800 μl, 13.3 x 8.7 cm (W x L), for use with Criterion and Criterion Dodeca cells
10x Tris/Glycine/SDS, Bio-Rad cat #161-0732, 1 L, 10x premixed electrophoresis buffer, contains 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 following dilution to 1x with water
SDS equilibration buffer II: 6M urea, 2% SDS, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, Bio-Rad cat #163-2108
And for the best results: Electrode Wicks, Bio-Rad cat #165-4071, Pkg of 500, precut electrode wicks, for use with the PROTEAN® i12™ IEF system. (I still have about 450, no need to buy)
2D PAGE video tutorial
2D PAGE detailed procedure for the Bio-Rad starter kit
Please let me know if you are interested in a 2D PAGE experiment!
