Faint Bands, High Background

EC887

A high background that obscures the bands, or in combination with fainter than usual bands, indicates dye binding to the gel matrix, or contamination of the matrix with a dye-binding material (most often a protein). Coomassie Blue R-250: Destaining time too short:  It takes hours for the dye to completely diffuse out of the gel…

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Faint bands, low background

EC890

SDS in the staining solution:  Most users save and re-use their staining solutions.  This is fine for a couple of cycles, but over time enough SDS elutes from the gels to accumulate to significant levels in the staining reagent.  SDS interferes with the binding of the dye to the proteins, resulting in bands that are…

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Coomassie Blue Stain- Troubleshooting

EC895

If your gel doesn’t look like this one, click on the problem below to find the solution: Faint bands on a low background Learn More Faint bands on a high background Learn More Uneven Staining Learn More Dark Blotches on Gel Learn More Smeared or blurred bands Learn More The ProtoGel Sample Prep Kit gives…

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Radioactive Emissions and the Use of Isotopes in Research

Radioactive decay occurs with the emission of particles or electromagnetic radiation from an atom due to a change within its nucleus. Forms of radioactive emission include alpha particles (α), beta particles (β), and gamma rays (γ). α particles are the least energetic, most massive of these decay products. An α particle contains two protons and…

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Ethidium Bromide Staining

Bands in gels stained with Ethidium Bromide fluoresce under ultraviolet light. The most commonly used stain for detecting DNA/RNA is ethidium bromide. Ethidium bromide is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm. Additionally, it can…

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Horizontal and Vertical Gel Systems – Vertical Tube Gels

Tube gels were used frequently in the development of gel electrophoresis. Although they are still used for some applications (most notably for isoelectric focusing as part of 2D electrophoresis), tube gels have been superseded by slab gels for most applications. Tube gels are cast (as the name implies) in glass tubes of 1-3mm diameter. The…

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Horizontal and Vertical Gel Systems – The Vertical Slab Gel System

A typical vertical apparatus used for sequencing is shown in the figure below. This system shows the components common to all vertical slab systems. The gel is cast between two glass plates, separated by spacers, typically less than 2mm thick. The gel is mounted in the system so that the top is in contact with…

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Horizontal and Vertical Gel Systems – The Horizontal Gel System

A gel electrophoresis apparatus must allow the researcher to maintain a uniform electric field across the gel, provide cooling to prevent thermal artifacts, and allow access to the gel for sample loading and monitoring the run. Two types of apparatus are in common use: vertical and horizontal. Vertical gel systems are further subdivided into slab…

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Buffer Additives-Reducing Agents

2-Mercaptoethanol

Disulfide bonds between or within sample protein molecules can lead to the formation of aggregates as well as play a role in the binding of the subunits of many proteins. It is usually desirable to cleave disulfide linkages prior to the protein electrophoresis. For this reason, disulfide bond reducing agents, such as 2-mercaptoethanol or dithiothreitol,…

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Buffer Additives-Surfactants

A crucial initial step in the electrophoretic separation of proteins is the solubilization of the sample molecules. This is especially true if there are extensive nonpolar interactions. Although urea in high concentration was often employed in the past for this purpose, researchers now often have recourse to the use of nonionic, anionic, or cationic detergents.…

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