Electrophoresis is a method used in western blot to separate and analyze proteins based on their size and charge. Electrophoresis can be one-dimensional (ie, one plane of separation) or two-dimensional. For most routine protein separations, one-dimensional electrophoresis suffices, while two-dimensional separation is used for more advanced proteomic studies in cells and involves isoelectric focusing in the first dimension.

Here we will focus on one-dimensional electrophoresis techniques. For those seeking a basic understanding of electrophoresis protocols for proteins, we recommend referring to the book "Gel Electrophoresis of Proteins: A Practical Approach" (Hames BD and Rickwood D, 1998, The Practical Approach Series, 3rd Edition, Oxford University Press).

Introduction to SDS-PAGE

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a widely used electrophoretic technique that separates proteins based on their molecular weight. By using sodium dodecyl sulfate (SDS) and polyacrylamide gel, this method eliminates the influence of protein structure and charge, enabling a focus solely on molecular weight differences.

SDS acts as a detergent, breaking down the tertiary structure of proteins and converting them into linear molecules for easier separation. Polyacrylamide, a chemically inert substance, is chosen for its versatility in creating gels with varying concentrations, resulting in different pore sizes that cater to specific separation needs. This flexibility allows researchers to tailor the conditions of SDS-PAGE according to their experimental requirements.

Preparation of PAGE gels

Polyacrylamide gels are essential components formed by the polymerization of acrylamide and N, N'-methylene bisacrylamide (or bis for short). The crosslinking agent for the gels is bis. Polymerization is initiated by adding ammonium persulfate (APS) along with either DMAP or TEMED. The gels are neutral and hydrophilic three-dimensional networks of long hydrocarbons cross-linked by methylene groups.

The separation of molecules within a gel is determined by the relative size of the pores formed within it. The pore size depends on two factors: the total amount of acrylamide present (%T) and the amount of cross-linker (%C). As the total amount of acrylamide increases, the pore size decreases. The smallest pore size is achieved with 5%C, while any increase or decrease in %C increases the pore size.

Gels can be purchased pre-made or produced in the laboratory using specific recipes found in laboratory handbooks.

It's crucial to carefully choose the percentage of acrylamide in your gel, as it determines the rate of migration and the degree of separation between proteins. For smaller proteins, you'll need a higher percentage of acrylamide and vice versa. Refer to Table 1 for guidance on selecting the appropriate gel percentage based on protein size.

Table 1. Protein sizes resolved by different gel percentages.

Note that acrylamide is a potent cumulative neurotoxin: always wear gloves when handling it.

For enhanced protein separation and resolution, consider using gradient gels. Unlike fixed-concentration PAGE gels, gradient gels have a continuous range of polyacrylamide concentrations, allowing for the resolution of a broader range of protein sizes on a single gel. Moreover, gradient gels yield sharper protein bands, facilitating better separation of similar-sized proteins and producing easily discernible data.

Once you prepared the suitable PAGE gel for your protein size, place the gel in the electrophoresis tank as instructed by the manufacturer and bathe in the migration (or running) buffer.

Molecular weight markers for electrophoresis

Molecular weight markers enable us to extrapolate the protein size of the sample (Figure 1) from the running characteristics of the marker and monitor the progress of an electrophoretic run. A range of molecular weight markers is commercially available. There are prestained markers and unstained markers that are both suitable for Western Blotting. The advantage of prestained markers is that they are also transferred to the Western Blot membrane and thus are more convenient in handling, while unstained markers require manual labeling as they are not visible without further staining.

Remember that the apparent molecular weight of markers can change depending on the running buffer chosen and the consequent pH of the system. The variance in pH between SDS-PAGE running buffers can affect the charge of the labeled protein standard and its binding capacity for SDS (demonstrated in Figure 1), causing a shift in mobility and an apparent change in molecular weight.

Figure 1. SDS-PAGE with prestained protein ladder – mid-range molecular weight (10–180 kDa) (ab116027) run with different SDS-PAGE buffer chemistries. Gel 1: Tris-Glycine (~4-20%), Gel 2: Bis-Tris (12%) MOPS buffer, Gel 3: Bis-Tris (10%) MES Buffer.

We have the following molecular weight markers:

Prism Ultra Protein Ladder (10-180 kDa) (ab116027)

Prism Ultra Protein Ladder (10-245 kDa) (ab116028)

Prism Ultra Protein Ladder (3.5-245 kDa) (ab116029)

Tips for loading samples and running the gel

To ensure accurate results, follow these tips when loading samples and running the gel:

• Include appropriate positive and loading controls in your experimental setup. See Recommended controls for western blot for more information.
• Use special gel loading tips or a micro-syringe to load the complete sample into wells.
• Avoid introducing bubbles during sample loading to prevent unequal loading. Load around 80% or less of the sample volume to avoid bubbles.
• Take care not to touch the bottom of the wells with the tip to avoid creating distorted bands.
• Avoid overfilling wells to prevent spillage into adjacent wells, which could lead to poor data and poorly resolved bands. When working with unique samples (eg, wild type vs knock-out), avoid loading samples in adjacent pockets to prevent spillover.
• Load 15–40 µg total protein per mini-gel well. If loading purified proteins, you might reduce the loaded amount.
• Submerge the gels in the running buffer (see a buffer recipe here), normally containing SDS, except in native gel electrophoresis.
• Run the gel for the recommended time as instructed by the manufacturer, as this can vary from machine to machine (eg, 30 minutes to overnight, depending on the voltage).
• When the dye (the migration front) reaches the bottom of the gel, turn the power off. Proteins will slowly elute from the gel at this point, so do not store the gel but proceed immediately to transfer.

Loading controls

Loading controls are required to ensure that the lanes in your gel have been evenly loaded with sample, especially when a comparison must be made between the expression levels of a protein in different samples. They are also useful to check for even transfer from the gel to the membrane across the whole gel.

Where even loading or transfer have not occurred, the loading control bands can be used to quantify the protein amounts in each lane. For publication-quality work, use of a loading control is absolutely essential.

The following table contains information about common loading controls:

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