Gel electrophoresis is a technique in which charged molecules are separated according to physical properties such as charge or mass as they are forced through a sieving gel matrix by an electrical current. Proteins are commonly separated in this manner using polyacrylamide gel electrophoresis (PAGE) to identify individual proteins in complex samples or to examine multiple proteins within a single sample.


Several forms of PAGE exist and can provide different types of information about the protein(s). Nondenaturing PAGE also called native PAGE, separates proteins according to their mass-charge ratio. Denaturing and reducing SDS-PAGE, the most widely used electrophoresis technique, separates proteins primarily by mass. Two-dimensional (2D) PAGE separates proteins by isoelectric point in the first dimenstion and by mass in the second direction.

SDS-PAGE separates proteins primarily by mass because the ionic detergent sodium dodecyl sulfate (SDS) denatures and binds to proteins to make them evenly negatively charged. Thus, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode. Proteins with less mass travel more quickly through the gel than those with greater mass because of the sieving effect of the gel matrix.

Once separated by electrophoresis, proteins can be detected in a gel with various stains, transferred onto a membrane for detection by Western blotting and/or excised and extracted for analysis by mass spectrometry. Protein gel electrophoresis is, therefore, a common step in many kinds of proteomics analysis.

Electrophoresis Technical Handbook

The Thermo Scientific Pierce Electrophoresis Tech Handbook provides information to improve the speed, convenience and sensitivity of your protein gel electrophoresis and staining applications. The Handbook covers all aspects of electrophoresis from sample and gel preparation to choice of molecular weight markers. In addition, it contains an extensive section on protein gel staining techniques and products.

Polyacrylamide gels

Acrylamide is the material of choice for preparing electrophoretic gels to separate proteins by size. Acrylamide mixed with bisacrylamide forms a crosslinked polymer network when the polymerizing agent, ammonium persulfate (APS), is added. TEMED (N,N,N,N'-tetramethylenediamine) catalyzes the polymerization reaction by promoting the production of free radicals by APS.

Polymerization and crosslinking of acrylamide. The ratio of bisacrylamide (BIS) to acrylamide, as well as the total concentration of both components, affects the pore size and ridgidity of the final gel matrix. These, in turn, affect the range of protein sizes (molecular weights) that can be resolved.
Example recipe for a traditional 10% mini gel for SDS-PAGE.
7.5mL 40% acrylamide solution
3.9mL 1% bisacrylamide solution
7.5mL 1.5 M Tris•HCl, pH 8.7
Add water to 30mL
0.3mL 10% APS
0.3mL 10% SDS
0.03mL TEMED

The size of the pores created in the gel is inversely related to the amount of acrylamide used. A 7% polyacrylamide gel has larger pores than a 12% polyacrylamide gel. Gels with a low percentage of acrylamide are typically used to resolve large proteins, and high percentage gels are used to resolve small proteins. "Gradient gels" are specially prepared to have low percent-acrylamide at the top (beginning of sample path) and high percent-acrylamide at the bottom (end), enabling a broader range of protein sizes to be separated.

Electrophoresis gels are formulated in buffers that provide for conduction of an electrical current through the matrix. The solution is poured into the thin space between two glass or plastic plates of an assembly called a "cassette". Once the gel polymerizes, the cassette is mounted (usually vertically) into an apparatus so that opposite edges (top and bottom) are placed in contact with buffer chambers containing cathode and anode electrodes, respectively. When proteins are added in wells at the top edge and current is applied, the proteins are drawn by the current through the matrix-slab and separated by the its sieving properties.

To obtain optimal resolution of proteins, a “stacking” gel is cast over the top of the “resolving” gel. The stacking gel has a lower concentration of acrylamide (e.g., 7% for larger pore size), lower pH (e.g., 6.8) and a different ionic content. This allows the proteins in a loaded sample to be concentrated into a tight band during the first few minutes of electrophoresis before entering the resolving portion of a gel. A stacking gel is not necessary when using a gradient gel, as the gradient itself performs this function.

Precast protein gels for SDS-PAGE. A Thermo Scientific Pierce Precise Protein Gel Cassette. The plastic cassette contains a mini-gel that is 1mm thick. Dividers along the top provide 10 wells for loading protein samples or molecular weight markers. Ordinarily, protein bands would not be visible until after electro-phoresis, disassembly of the cassette and staining of the gel. In this case, a stained gel image is superimposed on a cassette image for illustration.
Precast gels

Traditionally, researchers "poured" their own gels using standard recipes that are widely available in protein methods books. Most laboratories now depend on the convenience and consistency afforded by commercially available, ready-to-use, precast gels. Precast gels are available in a variety of percentages including difficult-to-pour gradient gels that provide excellent resolution and separate proteins over the widest possible range of molecular weights.

Technological innovations in buffer and gel polymerization methods enable manufacturers to produce gels with greater uniformity and longer shelf life than with traditional equipment and methods. In addition, precast polyacrylamide gels obviate the need to work with the acrylamide monomer – a known neurotoxin and suspected carcinogen.

Protein lanes and bands in 1D SDS-PAGE. Photograph of three mini-gels after removal from the cassette and staining with coomassie dye (Thermo Scientific GelCode Blue Stain Reagent). These mini-gels have ten lanes, each containing many protein bands of varying abundance.
1D Gel Electrophoresis

Comparative analysis of multiple samples is accomplished using one-dimensional (1D) electrophoresis. Gel sizes range from 2 x 3cm (tiny) to 15 x 18cm (large format). The most popular size (8 x 10cm) is usually referred to as a "mini-gel". Small gels require less time and reagents than their larger counterparts and are suited for rapid screening. However, larger gels provide better resolution and are needed for separating similar proteins or a large number of proteins.

Samples are added to sample wells at the top of the gel. When the electrical current is applied, the proteins move down through the gel matrix, creating what are called "lanes" of protein "bands". Samples that are loaded in adjacent wells and electrophoresed together are easily compared to each other after staining or other detection step. The intensity of staining and "thickness" of protein bands are indicative of their relative abundance. The position (height) of bands within their respective lanes indicates their relative sizes (and/or other factors affecting their rate of migration through the gel).

2D electrophoresis

Multiple components of a single sample can be resolved most completely by two-dimensional electrophoresis (2D-PAGE). The first dimension separates proteins according to their native isoelectric point (pI) using a form of electrophoresis called isoelectric focusing (IEF). The second dimension separates by mass using ordinary SDS-PAGE. 2D PAGE provides the highest resolution for protein analysis and is an important technique in proteomic research, where resolution of thousands of proteins on a single gel is sometimes necessary.

To perform IEF, a pH gradient is established in a tube or strip gel using a specially formulated buffer system or ampholyte mixture. Ready-made IEF strip gels (called immoblized pH gradient strips or IPG strips) and required instruments are available from certain manufacturers. During IEF, proteins migrate within the strip to become focused at the pH-points at which their net charges are zero. These are their respective isoelectric points.

The IEF strip is then laid sideways across the top of an ordinary 1D gels, allowing the proteins to be separated in the second dimension according to size.

Overview of 2D gel electrophoresis. In the first dimension (left), one or more samples are resolved by isoelectric focusing (IEF) in separate tube or strip gels. IEF is usually performed using precast immobilized pH-gradient (IPG) strips on a specialized horizontal electrophoresis platform. For the second dimension (right), a gel containing the pI-resolved sample is laid across to top of a slab gel so that the sample can then be further resolved by SDS-PAGE.

Native PAGE

In native PAGE, proteins are separated according to the net charge, size and shape of their native structure. Electrophoretic migration occurs because most proteins carry a net negative charge in alkaline running buffers. The higher the negative charge density (more charges per molecule mass), the faster a protein will migrate. At the same time, the frictional force of the gel matrix creates a sieving effect, retarding the movement of proteins according to their size and three-dimensional shape. Small proteins face only a small frictional force while large proteins face a larger frictional force. Thus native PAGE separates proteins based upon both their charge and mass.

Because no denaturants are used in native PAGE, subunit interactions within a multimeric protein are generally retained and information can be gained about the quaternary structure. In addition, some proteins retain their enzymatic activity (function) following separation by native PAGE. Thus, it may be used for preparation of purified, active proteins.

Following electrophoresis, proteins can be recovered from a native gel by passive diffusion or electroelution. In order to maintain the integrity of proteins during electrophoresis, it is important to keep the apparatus cool and minimize the effects of denaturation and proteolysis. Extremes of pH should generally be avoided in native PAGE as they may lead to irreversible damage to protein of interest, such as denaturation or aggregation.

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In SDS-PAGE, the gel is cast in buffer contain sodium dodecyl sulfate (SDS) and protein samples are heated with SDS before electrophoresis so that the charge-density of all proteins is made roughly equal. Heating in SDS, an anionic detergent, denatures proteins in the sample and binds tightly to the uncoiled molecule. Usually, a reducing agent such as dithiothreitol (DTT) is also added to cleave protein disulfide bonds and ensure that no quaternary or tertiary protein structure remains. Consequently, when these samples are electrophoresed, proteins separate according to mass alone, with very little effect from compositional differences.

When a set of proteins of known molecular weight are run alongside samples in the same gel, they provide a reference by which the mass of sample proteins can be determined. These sets of reference proteins are called molecular weight markers (MW markers) or standards, and they are available commercially in several forms. SDS-PAGE is also used for routine separation and analysis of proteins because of its speed, simplicity and resolving capability.

Watch this video on separating proteins by SDS-PAGE

Sample preparation reagents and loading buffers

Protein samples prepared for SDS-PAGE analysis are denatured by heating in the presence of a sample buffer containing 1% SDS with or without a reducing agent such as 20mM DTT, 2-mercaptoethanol (BME) or TCEP. The protein sample is mixed with the sample buffer and boiled for 3 to 5 minutes, then cooled to room temperature before it is pipetted into the sample well of a gel. Loading buffers also contain glycerol so that they are heavier than water and sink neatly to the bottom of the buffer-submerged well when added to a gel.

If a suitable, negatively charged, low-molecular weight dye is also included in the sample buffer, it will migrate at the buffer-front, enabling one to monitor the progress of electrophoresis. The most common tracking dye for sample loading buffers is bromophenol blue. Thermo Scientific Lane Marker Sample Buffers contain a bright pink tracking dye.

Samples may contain substances that interfere with electrophoresis by adversely affecting the migration of protein bands in the gel. Substances such as guanidine hydrochloride and ionic detergents can result in protein bands that appear smeared or wavy. The Thermo Scientific Pierce SDS-PAGE Sample Prep Kit facilitates removal of these interfering components using a specialized affinity resin system. Methods such as this are much faster and than dialysis, ultrafiltration or acetone precipitation and the protein recovery is generally higher.

SDS-PAGE band profile of the Thermo Scientific Spectra Multicolor Broad Range Protein Ladder. Images are from a 4-20% Tris-glycine gel (SDS-PAGE) and subsequent transfer to membrane. Some protein molecular weight markers are unstained and can be seen only upon staining or detection via fluorescent or enzyme probes.
Protein molecular weight markers

To assess the relative molecular weights (sizes) of proteins in a gel, a sample containing several proteins of known molecular mass run alongside the test sample in one or more lanes of the gel. Such sets of known proteins are called protein molecular weight markers or protein ladders. A standard curve can be constructed from the distances migrated by each marker protein. The distance migrated by the unknown protein is then plotted, and the molecular weight is extrapolated from the standard curve.

Several kinds of ready-to-use protein molecular weight (MW) markers are available that are labeled or prestained for different modes of detection. These are pre-reduced and, therefore, primarily suited for SDS-PAGE rather than native PAGE. MW markers are detectable via their specialized labels (e.g., fluorescent tags, see figure) and by ordinary protein staining methods.

Protein gel stains

Once protein bands have been separated by polyacrylamide gel electrophoresis, they can be blotted (transferred) to membrane for analysis by Western blotting (see related article) or they can be visualized directly in the gel using various staining or detection methods.

Coomassie dye is the most popular reagent for staining protein bands in electrophoretic gels. In acidic buffer conditions, coomassie dye binds to basic and hydrophobic residues of proteins, changing from dull reddish-brown to intense blue (see previous images on this page). As with all staining methods, Coomassie dye reagents detect some proteins better than others based on their chemistry of action and differences in protein composition. For most proteins, however, Coomassie dye reagents detect as few as 10 nanograms per band in a mini-gel. Thermo Scientific GelCode Blue and Imperial Stains use Coomassie G-250 and R-250 dyes, respectively.

Most staining methods involve some version of the same incubation steps:

  • A water-wash to remove electrophoresis buffers from the gel matrix
  • An acid- or alcohol-wash to condition or fix the gel to limit diffusion of protein bands from the matrix
  • Treatment with the stain reagent to allow the dye or chemical to diffuse into the gel and bind (or react with) the proteins
  • Destaining to remove excess dye from the background gel matrix

Another popular method for detecting protein bands within a gel is silver staining, which deposits metallic silver onto the surface of a gel at the location of protein bands. Commercial silver stain kits are exceptionally robust and easy to use, detecting less than 0.5 nanograms of protein in typical gels. Silver stains use glutaraldehyde or formaldehyde as enhancers, and typical formulations chemically crosslink proteins in the gel matrix. This limits the efficiency of destaining and recovery of proteins for downstream applications, such as mass spectrometry (MS). Thermo Scientific Silver Stain is fully compatible with destaining and elution methods required for MS analysis.

Another staining method compatible with protein recovery and mass spec analysis is the Pierce Zinc Reversible Stain. The Zinc stain is unique in that it does not stain the protein directly, but instead results in an opaque background with clear, unstained protein bands. The bands can be photographed by placing a dark background behind the gel. Zinc staining is as sensitive as typical silver staining (detects < 1ng of protein) and is easily erased, allowing trouble-free downstream analysis by mass spectrometry or Western blotting.

In recent years, the demand for fluorescent stains has increased with the improvements and popularity of fluorescence imaging equipment. Fluorescent stains are now available with excitation and emission maxima corresponding to the common filter sets and laser settings of most fluorescence imagers. Thermo Scientific Krypton Stains are state-of-the-art fluorescent protein stains.

Finally, several traditional and innovative chemistries exist for staining specific classes of proteins in polyacrylamide gels. These include stain kits to detect glycoproteins or phosphoproteins.

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  • Tech Tip #50: Process stained polyacrylamide gel pieces for mass spectrometry

Recommended reading
  • Coligan, J.E., et al. , Eds. (2002). Electrophoresis, In Current Protocols in Protein Science, pp. 10.0.1-10.4.36. John Wiley and Sons, Inc. New York.
  • Bollag, D.M., Rozycki, M.D. and Edelstein, S.J. (2002). Protein Methods, 2nd ed. Wiley-Liss, Inc. New York.
  • Hames, B.D. and Rickwood, D. Eds. (1990) Gel Electrophoresis of Proteins: a Practical Approach, 2nd ed. Oxford University Press, New York.