Tag Archives: 2D PAGE


Which reagent is better for reducing disulfides, DTT or TCEP? The two reagents are quite different in their reactivity, stability towards oxidation, reaction mechanisms, and other categories.


DTT is a thiol-containing reagent, and this must be considered in applications involving thiol labeling. TCEP is charged in solution and should not be used in isoelectric focusing. Aqueous solutions of TCEP are quite acidic (pH 2-3).

TCEP HCl is odorless, air-stable crystalline solid, soluble in water at a > 1 M concentrations. It reduces disulfides at room temperature in < 5 min in dilute solutions (5 -50 mM). There is no need to remove TCEP prior to the use of sulfhydryl-reactive labels or crosslinkers. TCEP is selective toward disulfides, and is reactive at a broad pH range.

For those who love Chemistry:

TCEP mechanism

Reduction of disulfide with TCEP. First step is rate-determining, kinetic rather than thermodynamic control.



Reduction of disulfide with DTT. Formation of stable cyclic disulfide drives the reaction.

You can learn more about DTT and TCEP from this article: “A Comparison between the Sulfhydryl Reductants Tris(2-carboxyethyl)phosphine and Dithiothreitol for Use in Protein Biochemistry”, Elise Burmeister Getz et al.  Analytical Biochemistry 273, p. 73–80 (1999)

2D PAGE in less than one page

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.


Immobilized pH gradient (IPG) strips

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!

Gel imaging equipment give-away

We are giving away a set of gel-imaging equipment. There has been a very small number of requests for use of this equipment even though we offer this service at no charge. We would like to find a good home for this equipment – hopefully a shared facility – as we are making room for a new mass spectrometer.

1. Proteome Works spot cutter with fluorescent enclosure (Bio-Rad)

IMG_1376 (427x640)





2. GS-800 calibrated densitometer (Bio-Rad)

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3. Molecular imager FX Pro Plus laser scanner (Bio-Rad)

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4. PDQuest server (Bio-Rad)

Please feel free to stop by and take a look!

1D and 2D PAGE equipment and supplies

I get a lot of inquiries about our PAGE (polyacrylamide gel electrophoresis) equipment, so here are some information on the equipment we have and the supplies we use.

Cost: It’s free!

We share our PAGE equipment with you at no charge. You need to purchase your own supplies: e.g. gels, standards, buffers, and other consumables. Sometimes we have supplies left over from the Facility experiments, which we will gladly share with you as well. Please, ask before you buy your own!


Power supply

Our power supply is a basic model; it does not store programmed methods. It can run up to 4 cells simultaneously, as long as the conditions are the same.

Electrophoresis cells

We have three Bio-Rad electrophoresis cells, a Mini-PROTEAN Tetra for up to 4 mini-gels (8.6 x 6.7 cm), a Criterion cell for up to 2 midi-gels (13.3 x 8.7 cm), and a Criterion Dodeca cell for up to 12 midi-gels. We also have a blotter for 13.3 x 8.7 cm gels.

2D PAGE, first dimension (isoelectric focusing, IEF)

Our PROTEAN IEF cell (Bio-Rad) is capable of storing methods, and accepts 7 cm, 11 cm, and 18 cm focusing trays. We also have rehydration trays for all three IPG (immobilized pH gradient) strip lengths.

Gel casting equipment

You can use our gel casting stands and glass plates (0.75 mm) to cast your own mini-gels. In my experience, 1-mm thick gels hold up much better, especially for low %T (4-10%). Bring your own plates if you wish!


The Pierce precast mini gels from Fisher are compatible with the Bio-Rad Mini-PROTEAN cell. We do not buy precast gels from Bio-Rad due to the high shipping costs. Fisher orders ship free. You can learn more here.

Immobilized pH gradient (IPG) strips are also available from Fisher (GE Healthcare Immobiline DryStrip gels, for example). The prices are comparable with the Bio-Rad’s, and the shipping is free.

05/28/2014 ETA: According to the GE Healthcare website, Immobiline IPG strips and buffers are not compatible with the Bio-Rad IEF cell running conditions. I am not familiar with the GE system and cannot tell you whether this is true; so to be safe, please use Bio-Rad IPG strips and buffers. If anyone has successfully run Immobiline strips on Bio-Rad IEF cell, please share with us! This information could save your colleagues both time and money!

Disclaimer: We do not receive any form of compensation for promoting any brand or a particular distributor.

Still have questions? Let’s meet and talk about your project!