Polyacrylamide Gel Electrophoresis

This article discusses the basics of polyacrylamide gel electrophoresis, including how it works, how the equipment functions and its various applications. To follow this article, as basic understanding of protein biochemistry is helpful.

Contents

Introduction

Polyacrylamide gel electrophoresis (PAGE) is a technique use almost universally in life science laboratories. The goal of this technique is to separate a mixed sample of proteins to identify and quantify single proteins from the mixture. The starting sample could come from any number of sources such as a patient sample, homogenised tissue or bacterial culture. It is also possible to use PAGE to separate DNA and RNA, but proteins are the most common sample type.
PAGE is often combined with a technique called western blotting. Blotting uses antibodies to bind to specific protein antigens, and so allows us to identify individual proteins with high specificity.

How does it work?

There are several types of PAGE technique that are used, but the most common is called SDS-PAGE. In SDS-PAGE the detergent Sodium dodecyl sulfate is used to denature the proteins and normalise their mass-to-charge ratio. Without SDS, both the molecular weight and the charge of the protein would affect its separation in the gel. With SDS, only the molecular weight affects the migration speed and so samples separate according to this. PAGE without SDS is called native PAGE, as the proteins stay in their native conformation.

The Gel Matrix

As with agarose gel electrophoresis, the samples are separated using an electrical field, and pass through a gel matrix which influences the migration of the proteins. In PAGE, rather than agarose, we use a chemical called polyacrylamide. Varying the percentage of polyacrylamide in the gel lets us change the size of the pores in the gel, which means that we can separate different sizes of protein in different percentage gels. Typical gel percentages are shown in the table below.

Acrylamide Percentage Separating Resolution
5 % 60 – 220 Kd
7.5 % 30 – 120 Kd
10 % 20 – 75 Kd
12% 17 – 65 Kd
15 % 15 -45 Kd
17.5% 12 – 30 Kd

Acrylamide is normally sold in a liquid form, as the powder form is neurotoxic an dangerous to handle. Polymerisation is achieved by mixing acrylamide with bis-acrylamide, which allows cross-links to form between the acrylamide molecules. Additional chemicals are added to initiate the polymerisation, usually ammonium persulphate as a source of free radicals and TEMED as a stabiliser. Once the polymerisation begins the gel is poured between 2 glass plates and allowed to completely polymerise. The gel mixture is made up not in water but in electrophoresis buffer (Tris-HCl), that provides the ions for electrophoresis. Often, the gel is poured in 2 parts. The first parts is a resolving gel, with a pH around 8.8 which slows the migration of the proteins. Above the resolving gel, a stacking gel is poured with a pH of 6.8 and a larger pore size. This stacking gel works to compress the protein samples into a thin migration front, so that all the proteins in the sample arrive at the resolving gel at the same time, leading to an accurate relative migration.

Running the gel

Unlike in agarose gel electrophoresis, where the gels are cast in trays are run horizontally, SDS-PAGE gels are cast vertically using a casting apparatus. we cast the gels in this way so that the stacking and resolving gels form a continuous gel, which would be much more difficult in a horizontal gel. It also allows a much greater protein amount to be loaded onto the gel.  The gel tank is also split into 2 sections. Depending on the manufacturer the tank will have an inner (or upper) buffer chamber, and an outer (or lower) buffer chamber. These two chambers are linked by the gel to create a continuous circuit. Each chamber contains and electrode, negative in the inner chamber and positive in the outer chamber. The inner chamber contacts the top of the gel, and when an electrical field is applied, the proteins will mode towards the positive electrode in the outer buffer chamber, due to the negative charges of the SDS molecules. Typical buffers for SDS-PAGE are Tris-Glycine for the buffer chambers, and Tris-HCl for the gel.

The speed of movement through the gel is then determined by the voltage gradient, i.e. the voltage between the electrodes. The required field strength is related to the size of the gel tank being used and the required voltage can be calculated using the simple equation E = V/d where E is the field strength, V the voltage and d the distance in cm between electrodes. Vertical gel tanks are generally run at 5 – 10 V / cm so if your tank has an electrode distance of 10 cm, you would run the gel at 50 – 100V. The exact value depends on your samples and should be determined empirically.

To apply this electrical field, we use a DC power supply. Most electrophoresis power supplies can be set to provide either a constant current or a constant voltage, with each having advantages and disadvantages. One potential issue is the production of heat due to the flow of current through the system which can be especially high with larger tanks that require higher voltage. For this reason, it is advisable to use some form of cooling, either passive in the form of a cooling block, or active such as a recirculating chiller, for larger electrophoresis systems.

Visualising the Protein

After migration, proteins must be visualised to determine their length and abundance. There are several methods commonly used to visualise proteins that are either specific or non specific. Non specific protein visualisation targets all proteins, using dyes that bind to common regions of the proteins such as the amino groups. Examples of non-specific protein stains include Coomassie Brilliant Blue and Ruby Pro. These stains are often non-reversible and can (but don’t always) interfere with downstream applications. Non-specific staining can be useful for quickly quantifying samples in a gel, or for ensuring a sample of interest is present.

To specifically visualise certain proteins, we need to use antibodies. Antibodies recognise unique 3 dimensional structures in the protein to distinguish them from others. By conjugating the antibody with a dye or enzyme, we can visualise just the protein that it recognises. The process of moving proteins from a gel to a membrane that can be probed with antibodies is called western blotting. To Learn more about western blotting, you can read our dedicated article soon. Whether you are western blotting or just non-specific staining, you will need to visualise the proteins using the gel documentation system. The type of system you use will vary based on whether you are using a visible stain like coomassie, or fluorescent or chemiluminescent dye attached to an antibody. As with agarose gel documentation systems, gel documentation systems for proteins can come in a variety of specifications depending on the requirements. For more advice on gel docs you can read our dedicated article.

Video Demonstration

Applications

SDS-PAGE and other forms of polyacrylamide gel electrophoresis are widely used in academic research into cellular and molecular biology. The ability to separate, identify and quantify the levels of proteins in certain cells and environments is essential for understanding how cellular processes work. Because of the ubiquity of these techniques any workflow that involves proteins is likely to include them, from a simple check of total protein content, all the way to whole proteome quantification using mass spectrometry.

Equipment for Polyacrylamide Gel Electrophoresis

Vertical Electrophoresis Tank Diagram
Components of the omniPAGE mini polyacrylamide gel electrophoresis tank

Gel Tank/Gel Box

The gel tanks used in vertical electrophoresis/SDS-PAGE differ from agarose gel tanks in a number of ways. As polyacrylamide gels are run in a vertical orientation, the gel tank include a module to hold the glass plates upright. Most modern PAGE tanks will use the glass plates to create the inner buffer chamber, by clamping them against a U shaped gasket opposite on another, thereby creating a section between the two plates that is separated from the rest of the chamber by the gel. The inner running module sits inside the gel tank and a lid is then connected which joins the tank to the power supply via the electrodes.

Cleaver Scientific manufactures a range of sizes of polyacrylamide gel tanks, all which compatible casting systems and western blotting modules. Our popular omniPAGE mini tank is compatible with all commercially available precast gels, making it ideal for fast routine protein separations.

Power Supply

To apply an electrical field to the gel, you will need an electrophoresis power supply. These power supplies are specifically manufactured for electrophoresis applications and features very stable voltage and current outputs to prevent fluctuations in migrations speed. A good power supply with allow you to set either constant current or voltage depending on the requirement of the experiment, and more advanced supplies will allow programming of individual steps at different parameter values.

At Cleaver scientific we have a range of electrophoresis power supplies for all applications. The PowerPRO series of power supplies is a versatile range designed to power both multiSUB horizontal and omniPAGE vertical electrophoresis tanks. Each power supply has a 2.4″ LCD display. Constant voltage, current and power options are available as well as pre-programmed or customer programmed conditions allowing users to save and repeat their experiments for exceptional reproducibility.

Gel Documentation System

For the final stage of the technique, gel imaging, you will need a gel documentation system as described above. Cleaver Scientific have a whole range of gel documentation systems to suite any budget or requirement. You can see our basic gel docs in the agarose gel electrophoresis article, or browse our western blot imagers below:

Reagents for Polyacrylamide Gel Electrophoresis

To run a gel electrophoresis experiment you will require both the equipment and the reagents. The basic reagents required for polyacrylamide gel electrophoresis are:

  • Acrylamide, TEMED and APS for making gels
  • Buffer stocks to make the running buffer
  • Loading dye to mix with Protein Samples
  • Protein Ladders to compare protein size and quantity
  • Protein stain for visualising protein

Cleaver Scientific supply many of these reagents: