Electrophoresis and Blotting

Within our Biometra product familiy we offer a comprehensive range of instrumentation for gel electrophoresis. Section Polyacrylamide Gel Electrophoresis (PAGE) contains numerous instruments for separation of proteins and related techniques.

For Agarose Gel Electrophoresis various systems for separation of DNA and RNA are offered. Suitable power supplies for the electrophoresis systems are available as well. In the Blotting section we offer various instruments for the transfer of proteins and nucleic acids from polyacrylamide gels, agarose gels or solutions onto carrier membranes (blotting membranes) like instruments for semi-dry and tank-blotting are available.

Electrophoresis and Blotting

Electrophoresis and blotting are two frequently combined methods in molecular biology. Combining both methods makes it possible to separate certain molecules from a mixture (electrophoresis) and subsequently transfer them to a membrane (blotting). In the final detection step, an antibody or a radioactively labeled probe can be added to the blot membrane to identify a molecule of interest. This makes it possible to reliably analyze a sample for nucleic acids or certain proteins.

The combination of electrophoresis and blotting is one of the most common methods in molecular analysis today. Typical applications include, for example, the detection of antibodies in blood serum as a diagnostic method for certain infectious diseases or the detection of gene sequences in a DNA mixture. Applications range from pharmaceuticals and biochemistry to medical diagnostics and research.

Origin and History of Electrophoresis and Blotting

The term electrophoresis refers to the spatial separation of charged molecules or groups of molecules in an electric field. The scientists Pyotr Ivanovich Strachov and Ferdinand Friedrich von Reuss succeeded in demonstrating the phenomenon of electrophoresis back in the early 19th century. However, it would be some time before electrophoresis could be used as a method in molecular biology: In 1937, the Swedish chemist Arne Tiselius developed a method for separating colloids in a carrier fluid by means of an electric field. His work represented the cornerstone of electrophoresis, also earning him the Nobel prize in chemistry in 1948. Electrophoresis underwent further developments as early as the 1940s, whereby solid phases were used for better separation. Over time, hydrogels such as agarose and polyacrylamide became established.

Seen historically, the evolution of blotting methods builds on the insights from electrophoresis research: In 1975, the researcher Edwin Southern succeeded in developing a blotting technique for DNA. This technique was named the "Southern blot" after its discoverer. In the subsequent years, new "blots" were developed, their names being plays on the original blotting technique, such as the "northern blot" for RNA analysis or the "western blot" for protein analysis. These blotting methods are used today in combination with gel electrophoresis.

Different gels can be used depending on the goal of the analysis. Agarose- or polyacrylamide-based gel electrophoresis is widely used:

• Agarose: Agarose gel features a wide-pored structure and is therefore ideal for separating nucleic acids. It behaves as follows: The more concentrated the agarose, the tighter-knit the matrix. A one-percent gel, for example, has a pore size of 150 nm, while a 0.16-percent gel has 500 nm pores. To create the characteristic sorted band pattern, a DNA-binding dye is usually added to the gel.
• Polyacrylamide: Compared to agarose gels, polyacrylamide-based gels are characterized by a significantly tighter structure with pore sizes between 3 and 6 nm; here, too, the pore size varies depending on the acrylamide concentration. Polyacrylamide gel electrophoresis (PAGE) is suitable for proteins with molecular masses between 1 and 500 kDa. Different variants have emerged from this method, of which SDS PAGE is one established method. In this method, sodium dodecyl sulfate (SDS) is added, which masks the intrinsic charge of proteins and linearizes the previously folded proteins.

One of the most important prerequisites for electrophoresis is the differing electrophoretic mobility of the particles being separated. Electrophoretic mobility is a measure of the migration speed of a particle in the gel. It is determined by various physical factors. In theory, these include the strength of the electric field, the size of the particle and the viscosity of the gel. In practice, other influencing factors such as the dissipative effect and the degree of dissociation of the electrolyte also play a role. 

If the electrophoretic mobility of the particles to be separated is different, a characteristic, size-dependent sorting of the molecules occurs during gel electrophoresis. The next step, blotting, involves transferring this sorting to a membrane. To do this, the material being investigated is transferred to a PVDF, nitrocellulose or nylon filter and fixed in place. This procedure allows the user to identify the molecule of interest by adding an antibody or a radiolabeled probe.

Like with electrophoresis, there are also different variants of blotting that are suitable for different applications. One of the most common methods in protein analysis is the western blot, which is suitable for qualitative and semi-quantitative detection of proteins. This method requires a membrane to be placed with its anode side on the electrophoresis gel and then placed between wet filter papers on both sides. Re-applying an electric field transfers the proteins to the matrix without losing the electrophoretically separated pattern. The sodium dodecyl sulfate (SDS) attached to the proteins can now be washed out.

In next step of protein detection, the temporary staining of all the proteins makes it possible to identify specific proteins, and to semi-quantitatively assess the amount of the protein.

Electrophoresis and Blotting Applications

The method that combines electrophoresis and blotting has become well established all across the pharmaceutical industry, medical diagnostics and biochemistry where it is among the most commonly used analytic methods for nucleic acids and proteins. The combination of gel electrophoresis and the western blot, for example, represents a proven method for detecting diagnostically relevant proteins, for instance when identifying antibodies in blood serum during an HIV test, or when searching for disease-related proteins in pharmaceuticals. 

In modern laboratory practice, the steps in the electrophoresis and blotting procedures can be designed efficiently and reliably using modular system solutions. For example, with its Biometra Eco-Line electrophoresis machine, Analytik Jena offers a system that optimizes PAGE electrophoresis protein analysis for daily use with a modular tank system and high-tech sealing material in the machine's gel casting stand. Devices such as the Biometra Fastblot are used for fast and homogeneous blotting of proteins and nucleic acids, reducing transfer times to a minimum and enabling reproducible, qualitative and quantitative protein analysis.