Biotin reveals itself to be a useful label for protein detection, purification and immobilisation because of its unique binding properties. It binds to avidin, streptavidin or NeutrAvidin proteins. This type of interaction is one of the strongest non-covalent interactions among protein-ligand interactions. Additionally, the small size of the molecule accounts for less interference with antibody function.

Biotinylation can be performed either by enzymatic or chemical methods, although chemical methods are most commonly used. Biotinylation reagents share the same overall structure:

  • the biotinyl group,
  • a spacer arm,
  • a functional group which reacts with the functional group of specific or non-specific amino acids within the target protein.

Common reactive groups and their respective targets on proteins include:

  • N-hydroxysuccinimide (NHS) and Sulfo-NHS – primary amines
  • maleimide, iodoacetyl groups or pyridyl disulfides – sulfhydryls
  • primary amines in combination with EDC – carboxyls
  • hydrazines and alkoxyamines – glycoproteins

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Enzymes are remarkable tools for the detection of proteins in tissues, whole cells or lysates. Various enzymes, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), can be attached to antibodies and proteins to act as chromogenic, chemiluminescent or fluorescent signal-generating molecules via their specific activity when put together with suitable substrates.

The high flexibility in signal output and amplification explains that enzymes are widely used as labels especially for antibodies. Multiple specific substrates are available for each enzyme allowing a which number of combinations. For example, our commercially available substrate for HRP, Covalight, which generates a stable chemiluminescent signal, is a perfect tool for Dot Blot and Western Blotting.

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Fluorescent probes

Fluorescent molecules also known as fluorophores are able to generate a detectable light signal in response to excitation by a source light beam at a specific wavelength. Unlike enzymes or biotin, fluorescent labels thus do not require additional reagents for detection. This characteristic makes fluorophores extremely versatile and therefore the new standard in detecting protein location and activation, identifying protein complex formation and conformational changes, as well as monitoring biological processes in vivo.

Available fluorophores inlude:

  • Organic dyes: Fluorescein isothiocyanate (FITC), Tetramethylrhodamine isothiocyanate (TRITC), DyLight® / ALEXA® Fluors and others ...
  • Quantum dots

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