Water for Protein Electrophoresis & Western Blotting

Section Overview

• What is protein electrophoresis?
• Impact of water quality in protein electrophoresis
• Overview of Western blotting: Protein transfer and detection
• Impact of water quality on Western blotting
• Study comparing various water qualities for Western blotting chemiluminescent detection 
• Ultrapure water for reliable, consistent protein electrophoresis & Western blotting
• Related Products

What is protein electrophoresis? 

Protein electrophoresis is used to separate proteins based on their size and charge. Since many important biological molecules, such as nucleic acids and proteins, have ionizable groups, they exist as electrically charged species at any given pH. As such, electrophoresis is one of the most widely used techniques in biochemistry and molecular biology.

Protein electrophoresis is simple, rapid and highly sensitive. It involves applying an electric field to a gel matrix through which proteins can migrate. The rate of movement of proteins through a gel is influenced by their charge, shape, and size. Smaller proteins generally move faster through the gel than larger ones because they navigate the pores in the gel matrix more easily. 

Types of protein electrophoresis

There are several types of protein electrophoresis, each serving specific purposes.

SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis is the most common method used to separate proteins primarily by size. SDS, a detergent, is used to denature and coat the proteins, giving them a uniform negative charge proportional to their length, thereby eliminating the influence of the protein’s natural charge.

Native PAGE: Unlike SDS-PAGE, native PAGE allows proteins to migrate through the gel in their natural states without being denatured. This method maintains the protein’s native shape and form, allowing for the separation based on both the protein’s charge and size.

Isoelectric Focusing (IEF): IEF separates proteins based on their isoelectric point (pI), the pH at which the protein has no net charge. Proteins are applied to a gel with a pH gradient and migrate until they reach the point in the gradient where their net charge is zero. This method has high resolution, being able to separate proteins that differ in their isoelectric points by as little as 0.01 pH unit.

2D Electrophoresis: Two-dimensional electrophoresis combines IEF and SDS-PAGE to provide a detailed analysis of complex protein mixtures. Proteins are first separated based on their isoelectric point and then by their molecular weight, resulting in a 2D separation pattern. Between 1000 and 3000 proteins from a cell or tissue extract can be resolved routinely using 2D-PAGE.

Once proteins are separated, they are commonly detected on gels using Coomassie Brilliant Blue, a sulfated trimethylamine dye. Silver staining is also used when greater sensitivity is required. Proteins separated on a gel may also be transferred to a membrane for Western Blotting, an immunoblotting technique used to probe the proteins that were previously separated in the electric field.

Impact of water quality in protein electrophoresis

Water is used in many steps of the gel electrophoresis process: for sample preparation, to prepare buffers, reagents and stains, and possibly also to prepare the gel. The quality of the purified water used can affect the accuracy of the results as follows:

Organics

Acrylamide gels are formed from the polymerization of acrylamide monomers in the presence of smaller amounts of bis-acrylamide (a cross-linking agent). Organic contaminants may affect the polymerization, resulting in poor quality gels.

Ions

The migration of proteins in the gel is dependent on, among other things, ionic strength and pH of the running buffer. The ionic strength and pH of buffers used to prepare the gel and running buffer have to be maintained to assure reproducible results. The presence of high levels of ions could alter the ionic strength of the solutions and this could affect the migration rate of proteins.

Bacteria

Water that is contaminated by bacteria could contain degradation by-products, such as proteases that may degrade proteins, and ions which, as previously mentioned, may alter the ionic strength of solutions.

Water quality recommendation

Pure (Type 2) water is recommended for protein electrophoresis. Ultrapure (Type 1) water can also be used, depending on sensitivity. A range of water purification solutions adapted to the needs of scientists performing protein research is available. 

 

Overview of Western blotting: Protein transfer and detection

Western blotting is a cornerstone technique in protein research. It is used extensively to detect and quantify proteins in complex mixtures. Western blotting starts by separating a protein sample by gel electrophoresis. Once separated in the gel, proteins are transferred or blotted to a membrane via electrophoresis (Figure 1). The gel and membrane are sandwiched between blotting papers soaked in buffer, and the set is compressed between two parallel electrodes in a cassette. Current is passed at right angles to the gel, causing the proteins in the gel to migrate towards the membrane.

Figure 1. Elements of a Western blot transfer assembly

General steps of Western blotting

Once the proteins have been transferred to the membrane (referred to as a blot), they can be detected by several methods, such as chemiluminescence, fluorescence, chromogenic, and autoradiography. The detection step usually involves probing the blot with an antibody to detect a specific protein following these general steps:

1. Blocking: The blot is incubated in a blocking solution [e.g. 5% (w/v) non-fat dry milk or 10% (w/v) bovine serum albumin] to block all remaining binding sites on the membrane.
   
   
2.  Probing with Primary Antibody: The blot is next incubated in a dilution of antiserum (the primary antibody) directed against the protein of interest.
   
   
3. Probing with Secondary Antibody: After several washing steps, the blot is incubated with a labeled secondary antibody, which is directed against the species that provided the primary antibody.
   
   
4. Detection: The label on the secondary antibody provides a way to visualize the interaction of the secondary antibody with the primary antibody on the blot. The label could be a fluorescent molecule, isotopic labels, and more commonly, enzymes. When an enzyme-labeled secondary antibody is used, the blot is incubated in a substrate which gets converted to a product that can be detected (e.g. light, colored precipitate).

Impact of water quality on Western blotting

In Western blotting, water is used to prepare the transfer buffer, and the buffers used to prepare the blocking solution, primary and secondary antibodies, and the wash solutions. To enhance protein detection, it is important to use water that is free of the following contaminants:

Ions

The stability of buffer ionic strength is important in the Western blotting phase to keep consistency from one run to another. The water used to prepare the buffers needs to have consistently low ionic contamination.

Bacteria and bacterial by-products

Biologically active molecules resulting from bacterial contamination will compromise the quality of Western blotting experiments. For instance, if the secondary antibody is labeled with alkaline phosphatase, the water used in Western blotting needs to be alkaline phosphatase-free to make sure that background chemiluminescence is kept at a minimum. In addition, bacteria can release proteins that could interfere with the blotting process.

Organics

Water that is grossly contaminated with organics could affect fluorescence signals in Western blotting detection steps. It is advisable to keep organic contamination to a minimum.

Study: Comparison of different water qualities for Western blotting chemiluminescent detection

Success in Western blotting relies not only on a good separation of proteins by gel electrophoresis and the subsequent efficient transfer of proteins from the gel matrix to a solid membrane support, but it also relies heavily on a highly sensitive and selective method to detect the proteins. In this experiment, different types of water were used in the preparation of gel electrophoresis running buffer, transfer buffer, blocking solution, primary and secondary antibodies, and for all the rinsing steps.

Water types affect chemiluminescent signals differently

Figure 2 shows a comparison of background chemiluminescent signals obtained when ultrapure, molecular biology (mol biol) grade bottled, deionized, or reverse osmosis (RO) water was used for all steps of Western blotting. The lowest background signal comes from freshly purified ultrapure water obtained from a Milli-Q^® ultrapure water system fitted with an ultrafiltration polisher (Biopak^®) installed at the point of use. This suggests that a better assay sensitivity can be obtained with ultrapure water.

Figure 2. Background chemiluminescence signals from different types of water purification.

Ultrapure water for reliable, consistent protein electrophoresis & Western blotting

Ultrapure (Type 1) water purified with an ultrafiltration final filter is highly desirable for buffer and gel preparation for electrophoresis and Western blotting to avoid contamination, improve protein separation, and increase sensitivity. A range of water purification solutions adapted to the needs of scientists working with protein electrophoresis and Western blotting is available.

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