Our Glossary

Two dimensional (2D) gel electrophoresis is a form of gel electrophoresis used to analyse complex proteins. Mixtures of proteins are separated by two properties in 2D gels by their isoelectric points and by their molecular weights.

Agarose gel electrophoresis is the most commonly used technique for separating nucleic acid fragments that offers a broad separation range. A wide range of different sized nucleic acid molecules can be detected by adjusting the concentration of the agarose gel. The DNA/RNA samples may be separated by charge and/or size. The migration of nucleic acids in agarose gels is also affected by the choice of running buffer and the applied voltage.

Bioluminescence is the production and the emission of light by a living organism. Reactions that cause bioluminescence commonly involve a chemical called luciferin. Luciferin interacts with luciferase and light is produced as a by-product.

The focal length of the lens is calculated from the distance from the optical centre of the camera lens to that of the image sensor. The focal length of the camera lens is normally expressed in millimetres e.g. 50mm.

A charged -coupled device (CCD) is a silicon based imaging device (chip) which converts photons of light into electron representatives.

Chemiluminescence is the generation of electromagnetic radiation as light by the release of energy from a chemical reaction. The most popular chemiluminescent western blotting substrates are luminol based and the commonly used reporters that catalyse chemiluminescent reactions are HRP and alkaline phosphatase (AP).

Cloning refers to the process by which recombinant DNA molecules are produced and transformed into a host organism, where they are replicated.

One method of determining the concentration of microbes in a sample is to dilute the sample, grow the microbes on plates and count the colonies. Colony counts are used to detect and count microbes in soil, water and food.

After transfer and before proceeding with the western blot, total protein on the membrane is often stained with a dye, such as Ponceau S or amido black 10B to check transfer efficiency.

Most dyes interfere with antibody binding and detection therefore, a protein stain that is easily removable is ideal such as Ponceau S.

Denaturing gradient gel electrophoresis (DGGE) is based on finger printing methodology. The gel is subjected to a constant heat of ~60⁰C and increasing concentration of denaturing chemicals are used to force DNA molecules to un-fold (melt) which are also known as melting domains. Any variation of DNA sequences within these domains will result in different melting temperatures causing different sequences to migrate at different positions in the gel.

DIGE is a 2D gel that uses direct labeling of proteins with fluorescent dyes (known as CyDyes: Cy2, Cy3 and Cy5) making it easy to multiplex different protein samples within the same 2D gel. This permits the inclusion of an internal standard within each gel, limiting the experimental variation and ensures accurate intra-and inters gel matching.

Each molecule of DNA consists of two strands coiled round each other to form a double helix. DNA consists of a pair of chemical groups called bases (there are four types G,C, A and T), which combine in specific pairs (G-C and A-T)  so that the sequence on one strand of the double helix is complementary to that on the other: it is the specific sequence of bases which codes amino acids which are the building blocks of proteins.

A lens typically has two measurements of F-stop (aperture). The maximum aperture is when the lens is fully open and the minimum aperture is just before the lens is completely closed. The F-stop has a number of effects upon the final image, a low f-stop e.g. f/0.95 the lens will be able to pass more light in dark conditions and a maximum f-stop e.g. f/14 may be necessary where there is a very high level of light.

Photographic film can be used with enhanced chemiluminescene (ECL) substrates for horseradish peroxidase (HRP) or alkaline Phosphatase (AP).

Fluorescence occurs when molecules (fluorophores) absorb light in their ground state. Fluorophores do not emit light but when exposed to a light source (excitation by UV, RGB and IR light) their energy levels are elevated to a breif but unstable excited state. As the fluorophore returns to its ground state it emits light at a lower energy which is at a longer wavelength than that of the excited light source.

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation.

Gel documentation systems are used to image labeled nucleic acid and protein samples in various types of media such as agarose, acrylamide or PVDF or nitrocellulose. Gel doc systems come in a variety of configurations depending on throughput and sample type.

Gel electrophoresis is a method for the separation and analysis of macromolecules (DNA, RNA and protein) based on size and charge. There are two different types of gels commonly used agarose and polyacrylamide gels.

1D electrophoresis is performed on nucleic acids and proteins where as, 2D electrophoresis is for separating protein based on size and pH.

Gel imaging systems are used to image labeled nucleic acid and protein samples in various types of media such as agarose, acrylamide or PVDF or nitrocellulose. Gel imaging systems come in a variety of configurations depending on throughput and sample type.

GFP is useful for directly monitoring gene expression and as a transformation marker in living plants. GFP is a protein that exhibits bright green fluorescence when exposed to blue or UV light. There are several derivate forms of GFP, enhanced (eGFP) and wild type (wtGFP) are the most commonly used.

The in-cell western (ICW) assay is an immunofluorescence assay typically performed in microtiter plates (96 and 384 well formats). This quantitative technique combines the specificity of western blotting with the reproducibility and the throughput of an ELISA.

In-Gel westerns permit the detection of protein directly in the polyacrylamide gel without having to transfer the gel to a membrane or carry out any blocking steps. In-Gel westerns can be used to detect single or multiple proteins on the same gel by using one or more fluorophore.

In-vivo imaging is an optical imaging technique which looks deep into tissues of living test subjects.  Near-infrared light is often used and a CCD camera optimised for NIR imaging.

Visible fluorescence can be limited due to high backgrounds from membranes and biological materials. Some proteins autofluoresce strongly in the visible light channels (blue, green and red) also membranes such as PVDF and nitrocellulose can also fluoresce under blue and green light. With Infra-red imaging, membrane and protein autofluorescence is negligible. Commonly used IR secondary antibodies are IRDyes 680 and 800 and DyLight 680 and 800.

A camera lens gathers and focuses the light reflected from an object.  The reflected light rays enter the camera lens and pass through the elements and are directed to the camera’s image sensor.

There are two main types of membranes PVDF (polyvinylidene difluoride) and nitrocellulose, both available in different pore sizes and dimensions depending on your application needs.

PVDF membranes are highly hydrophobic and must be pre-wetted with methanol. This type of membrane has a high binding affinity for proteins and nucleic acids and is often used for the following applications – western, southern, northern and dot blots.

Nitrocellulose membranes are popular in protein blotting due to its high-binding affinity and compatibility with a variety of detection methods including chemiluminescence, chromogenic and fluorescence.

Molecular weight size marker (ladder) is also referred to as a protein, DNA or RNA ladder. These ladders are a set of standards that are used to identify the approximate size of a molecule run on either an agarose or polyacrylamide gel during electrophoresis. The approximate size calculation is based on the principle that molecular weight is inversely proportional to the migration rate of the molecule through a gel.

Multiplexing fluorescent western blotting allows for the detection and quantification of multiple proteins simultaneously on the same sample. By using highly specific fluorescently labeled antibodies, direct protein abundance comparisons and normalisation against a housekeeping gene can be performed saving you time as you no longer need to strip and re-probe your blot.

Nitrocellulose membranes are popular in protein blotting due to its high-binding affinity and compatibility with a variety of detection methods including chemiluminescence, chromogenic and fluorescence.

Polymerase chain reaction (PCR) is used for a wide range of applications such as cloning, gene expression analysis, genotyping, sequencing and mutagenesis.

PCR is a technique that enables many copies of a specific DNA region to be amplified. PCR relies on thermostable DNA polymerase, Taq polymerase and requires DNA primers designed to specifically for the DNA region of interest.

Polyacrylamide is a popular choice for preparing electrophoretic gels when you need to separate proteins by size. Acrylamide is often mixed with bisacrylamide and can be prepared at different percentages depending on the size of proteins to be resolved. Low-percentage gels are used to resolve large proteins, and high-percentage gels are used to resolve small proteins.

Proteins are large molecules composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein. Proteins play an important role as they are required for the structure, function, and regulation of the body’s cells, tissues, and organs.

PVDF membranes are highly hydrophobic and must be pre-wetted with methanol. This type of membrane has a high binding affinity for proteins and nucleic acids and is often used for the following applications – western, southern, northern and dot blots.

‘Safe’ dyes are a safer alternative to Ethidium bromide which is a highly mutagenic material. ‘Safe’ dyes can be visualised using either UV or blue light.

Signal to noise ratio (SNR) of a CCD image sensor specifically represents the ratio of the measured light signal to the combined noise.

Stain Free imaging technology utilises a polyacrylamide gel containing a proprietary tri-halo compound to make protein fluoresce directly in the gel after activation of UV light, allowing the immediate visualisation of proteins on the gel.

Thin layer chromatography (TLC) involves a liquid sample migrating through capillarity through a solid adsorbent medium (such as alumina or silica gel) which is arranged as a thin layer on a rigid support (i.e. glass plate).

UV shadowing is a technique used to detect DNA/RNA and it involves the gel (polyacrylamide) being placed on a plastic wrap over a UV fluorescent TLC plate. The TLC plate contains a dye which is excited by UV light. Dark areas are observed where the DNA in the gel absorbs the UV light.

Western blotting is a core technique in cell and molecular biology. Western blotting is used to detect the presence of a specific protein in a complex mixture extracted from either cells or tissue.

This technique has three stages; the separation of protein mixtures by size using gel electrophoresis; the efficient transfer of separated proteins to a solid support such as a nitrocellulose or PVDF membrane; and the specific detection of a target protein by appropriately matched antibodies. Once detected, the target protein will be visualized as a band on a blotting membrane on an imaging system.