Order Entry
Introduction to SDS Page | Avantor

Introduction to SDS-PAGE – Separating Proteins Based on Molecular Weight

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, or SDS-PAGE, is a widely used technique for separating proteins based on their molecular weight. In this technique, proteins are treated with the detergent SDS, which binds to the proteins and denatures them, breaking down their quaternary and tertiary structures. Adding SDS also imparts a negative charge to the proteins, allowing them to be separated by size.

Proteins are next loaded into a polyacrylamide gel cast into a thin slab or a column. The polyacrylamide gel comprises two parts: the resolving gel and the stacking gel. The stacking gel has a lower acrylamide concentration and is placed above the resolving gel. This arrangement allows proteins to stack in a narrow band in the stacking gel before they enter the resolving gel.

This article focuses on the capabilities of SDS-PAGE, how it is implemented, and what are the possible applications of this technique.

Properties: What is SDS-page?

SDS-PAGE can help researchers obtain accurate and reliable results because of its ability to separate proteins based on their molecular weight, its high resolution, sensitivity and reproducibility.

These capabilities make SDS-PAGE a valuable tool for various applications, including protein identification, quantification and purity assessment. Here are some of the methods used to make SDS-PAGE a valuable tool:

Electrophoresis method

SDS-PAGE is a form of electrophoresis involving the movement of charged molecules in an electric field. Proteins are denatured and treated with the anionic detergent SDS, which imparts a negative charge proportional to their size.

Separation based on molecular weight

SDS-PAGE separates proteins based on their molecular weight by subjecting them to an electric field that causes them to migrate through a polyacrylamide gel matrix. Smaller proteins move faster through the gel, while larger proteins move more slowly.

Protein denaturation

In SDS-PAGE, adding SDS to a sample causes proteins to denature, breaking down their quaternary and tertiary structures. This allows for uniform binding of the negatively charged SDS molecules.

Gel matrix

The polyacrylamide gel matrix used in SDS-PAGE comprises the resolving and stacking gel. The stacking gel has a lower acrylamide concentration and is placed above the resolving gel. This arrangement allows proteins to stack up in a narrow band in the stacking gel before they enter the resolving gel, which helps to focus the protein bands and improve the separation resolution.


After electrophoresis, proteins can be visualized by staining the gel with a protein-specific dye, such as coomassie blue or silver stain. The stained gel can be analyzed to determine the molecular weight of the separated proteins.

Protocol – How does SDS page work?

SDS-PAGE comprises many processes that need to be completed consecutively. Here are the processes in the order they must be performed.

Gel preparation

The first step in SDS-PAGE is preparing the polyacrylamide gel matrix. The gel is prepared by  mixing the acrylamide monomers with a crosslinker, a polymerization initiator, and a buffer solution. The mixtrue is then poured into a casting tray and allowed to polymerize.

Polyacrylamide gels are used in SDS-PAGE because they provide a porous matrix through which proteins can migrate during electrophoresis. The matrix is created by polymerizing acrylamide monomers with a crosslinking agent, typically N, N'-methylene bisacrylamide (bis), in the presence of a free radical initiator such as ammonium persulfate (APS) and N, N,N', N'-tetramethylethylenediamine (TEMED). The acrylamide concentration of the gel is usually between 5% and 20%, depending on the size range of the proteins to be separated.

The acrylamide monomers form long chains crosslinked by the bis molecule to create a three-dimensional network. The resulting gel matrix has a defined pore size that determines the resolution of the separation. The pore size is determined by the ratio of acrylamide to bis in the gel, with higher ratios resulting in smaller pores and better resolution for smaller proteins.

The gel matrix is poured into a casting tray and allowed to polymerize. Once polymerized, the gel is removed from the casting tray and placed into the electrophoresis apparatus connected to an electrical power supply. The gel is then equilibrated in a running buffer, typically Tris-glycine buffer, before sample loading and electrophoresis.

Sample preparation

Before loading protein samples onto the gel, the sample must be mixed with a loading buffer containing SDS, reducing agents and tracking dyes. The reducing agents help break down disulfide bonds in the proteins while the tracking dyes allow researchers to monitor the migration of the proteins through the gel.

Protein samples for SDS-PAGE must first be denatured with a detergent, such as sodium dodecyl sulfate (SDS), to disrupt non-covalent interactions and to give the proteins a uniform negative charge density, which allows them to migrate towards the anode during protein electrophoresis.

Samples are typically treated with a reducing agent, such as beta-mercaptoethanol or dithiothreitol (DTT), to break disulfide bonds and prevent protein aggregation.

The protein samples are mixed with a loading buffer, typically containing SDS, a reducing agent, a tracking dye, and a buffer to adjust the pH. The tracking dye is a colored compound that migrates with the proteins during electrophoresis and allows the user to monitor the progress of the gel.

The sample is heated to 95-100°C for 5-10 minutes to denature the proteins and disrupt disulfide bonds fully. The heated sample is then loaded into the wells of the polyacrylamide gel using a pipette or syringe.

The amount of protein loaded into each well can be controlled by adjusting the sample volume and/or the concentration of the loading buffer. The protein sample is typically loaded into wells pre-cast into the gel, either in a single horizontal row or in a vertical stack.

SDS page electrophoresis

In SDS-PAGE electrophoresis, samples are loaded into the gel wells, and an electric field is applied across the gel to drive negatively charged proteins through the matrix. Proteins move through the stacking gel and then into the resolving gel, where they are separated based on size. That continues until the tracking dyes have migrated a certain distance, indicating that the proteins have been separated.

The polyacrylamide gel is placed in a buffer-filled electrophoresis apparatus, typically consisting of a tank with two electrodes, the anode and cathode. The gel is carefully placed into the buffer, and the electrodes are connected to a power supply.

When the current is turned on, negatively charged proteins move towards the anode that is usually positioned at the bottom of the gel. The speed of migration is determined by the size and charge of the protein, as well as the strength of the electrical field and the concentration of the polyacrylamide gel.

The electrophoresis procedure is typically run for 1-2 hours, depending on the size and number of separated proteins. The progress of the separation can be monitored visually by watching the tracking dye as it migrates through the gel.

Gel staining

After electrophoresis, separated proteins are visualized by staining the gel with a protein-specific dye, such as Coomassie blue or silver stain. The dye binds to the proteins, allowing them to be seen as distinct bands in the gel. This step is important for identifying and quantifying the separated proteins.

Coomassie Brilliant Blue is a dye that binds to the proteins in the gel and turns them blue. The dye is typically mixed with methanol and acetic acid solution, dehydrating the gel and allowing the dye to penetrate the proteins. After staining, the gel is typically destained with methanol and acetic acid solution, which removes excess dye and allows the protein bands to stand out more clearly.

Silver stain is more sensitive than Coomassie Brilliant Blue and can detect lower amounts of protein. The staining procedure involves several steps, including sensitization of the gel, impregnation with a silver solution and stain development. After staining, the gel is typically washed and fixed to preserve the stain.

After the gel is stained and de-stained, the protein bands can be visualized as distinct bands or spots on the gel. The protein bands can be compared to molecular weight markers that were run alongside the samples to determine the approximate molecular weight of each protein.

Reversible gel staining

In some applications, it may be necessary to extract proteins from the gel for further analysis, such as mass spectrometry. In this case, the gel staining can be reversed using a destaining solution that removes the dye from the proteins. The de-stained gel can then be used for protein extraction or further analysis.

Each of these steps is critical to the success of the SDS-PAGE procedure, and researchers must carefully optimize each step for their specific application to achieve accurate and reproducible results.

Molecular weight determination 

Molecular weight determination is a key aspect of SDS-PAGE. In this technique, the migration of protein molecules through the gel is influenced by their size and charge.

As proteins move through the gel, they encounter a meshwork of polyacrylamide, which acts as a molecular sieve. Smaller proteins can move more easily through the gel matrix, while larger proteins experience more resistance and move more slowly.

To determine the molecular weight of a protein using SDS-PAGE, a series of protein standards with known molecular weights are typically run alongside the unknown protein sample. These standards are proteins of a known size run on the same gel as the sample, and their migration through the gel can be used to calibrate the gel and determine the size of the unknown proteins.

After the gel has been run, the protein bands are visualized using staining methods such as Coomassie blue or silver stain. The relative migration of the protein bands is then compared to that of the protein standards, which have been run in parallel. This allows the molecular weight of the unknown proteins to be estimated based on their migration relative to the standards.

Applications of SDS-page

SDS-PAGE is a widely used technique with various applications in biochemistry, molecular biology and related fields. One of the most common uses of SDS-PAGE is to analyze protein samples for purity and size. By running protein samples on an SDS-PAGE gel, researchers can separate individual proteins based on their molecular weight and confirm the identity and purity of the protein of interest.

SDS-PAGE is also frequently used to isolate and purify proteins from complex mixtures. After running a sample on an SDS-PAGE gel, the protein band of interest can be excised and the protein extracted for further analysis.

In addition, SDS-PAGE is used in various other applications, including protein identification and quantification, characterization of post-translational modifications, and determination of protein-protein interactions. Researchers in academia, industry and government labs worldwide use the technique.

Learn more about SDS-PAGE

You can learn more about SDS-PAGE and some of the products available to support the technique.