Protein Tags

The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria which fluoresces green upon excitation with blue or ultraviolet light. The discovery of green fluorescent protein in the early 1960s by Shimomura has laid the foundation for modern live cell imaging. GFP can be fused to other proteins of interest in a host cell as a noninvasive fluorescent marker in living cells and organisms. Since then, GFP has been engineered and further developed to produce a plethora of mutants coverving a wide range of colors, including enhanced green, cyan and yellow fluorescent proteins, which exhibit peak emission wavelengths ranging from 425 to 525 nm.

Learn more about protein tags down below and discover high quality antibodies against fluorescent protein tags. antibodies-online offers GFP catchers, the ideal tool for effective pulldown of GFP fusion proteins. Our catcher product line is based on recombinant single-domain antibodies (sdAbs) derived from llamas or alpacas. They are the ideal tool for effective pulldown of fusion proteins.

Comparison of GFP Variants

Because the construction of red-shifted mutants from the Aequorea victoria jellyfish GFP beyond the yellow spectrum proved largely unsuccessful, researchers have searched for additional options. In 1999 the red fluorescent protein DsRed, derived from Discosoma sea anemones was mentioned first in a publication by Matz et al. Once fully matured, the fluorescence emission spectrum of DsRed features a peak at 583 nm whereas the excitation spectrum has a major peak at 558 nm and a minor peak around 500 nm.

Fluorescent Protein Tags: GFP vs RFP

In comparison to GFP, wild-type DsRed shows certain downfalls but also some unique benefits. Unlike GFP, the fluorescence of DsRed remains stable in acidic pH ranges. Furthermore, DsRed is particulary well suited for fluorescence microscopy, due to its good contrast. Additionally, it has a high quantum yield and is photo stable. On the other hand, maturation of DsRed fluorescence occurs slowly over a time frame of 24 hours. Thus, it cannot be used to monitor proteins with a short halflife. DsRed is an obligate tetramer and can form large protein aggregates in living cells which pose a higher risk for toxicity. Overall, GFP can successfully conjugate with a broader variety of proteins.

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antibodies-online offers catchers, the ideal tool for effective pulldown of GFP, RFP or BFP fusion proteins. Our catcher product lines are based on recombinant single-domain antibodies (sdAbs) derived from llamas or alpacas. This type of affinity molecules provide significant advantages over conventional IgG molecules. All of our products are produced in-house. They are highly specific with affinities tailored for a wide range of applications.

RFP Variants

Similar to GFP several RFP variants were engineered to improve its characteristics in general or optimize for specific experiments. DsRed2 contains several mutations at the N-terminus that prevent formation of protein aggregates and reduce toxicity. Red fluorescence emission from DsRed-Express can be observed within an hour after expression, 11 times faster than DsRed. DsRed-Express is a precursor RFP of more recent derivatives including tdTomato and the monomeric mCherry, mOrange, and mStrawberry.

Comparison of RFP Variants

mCherry is together with mOrange and mStrawberry a member of the mFruits family of monomeric red fluorescent proteins. In comparison to the progenitor DsRed, mOrange, mStrawberry and mCherry produces strong blue- and red-shifted variants. They have a lower molecular weight and will fold faster than tetramers do and therefore disturb the target system less. Out of all of the true monomers developed, mCherry has the longest wavelengths, the highest photostability, fastest maturation and excellent pH resistance. mOrange overcomes mCherry in quantum yield however it does exhibit substantial acid sensitvity. A high extinction coefficient and quantum yield makes the large stoke shift variant LSSmOrange (in combination with T-Sapphire) a suitable candidate for FRET applications.

tdTomato is a genetic fusion of two copies of the dTomato gene which was specifically designed for low aggregation. Its tandem dimer structure plays an important role in the exceptional brightness of tdTomato, which is 5 times higher compared to EGFP. tdTomato's emission wavelength (581 nm) and brightness make it ideal for live animal imaging studies. Because tdTomato forms an intramolecular dimer, it behaves like a monomer and is as photostable as mCherry.

Phototransformable Fluorescent Proteins

Dendra2 is an improved version of the green-to-red photoswitchable fluorescent protein Dendra, derived from octocoral Dendronephthya species. Dendra2 exhibits faster maturation and brighter fluorescence both before and after photoswitching than that of Dendra. In contrast to all other monomeric photoactivatable FPs, which necessarily require UV-violet (e.g., 405 nm laser) light for activation, Dendra and Dendra2 permit the use of blue (e.g., 488 nm laser) activating light. The phototransformative property has allowed highlighting and tracking of subpopulations of cells, organelles, and proteins in living systems. With Dendra2 newly synthesized proteins that are en route to their final destinations can be visualized.

Protein tags are various, mostly short, amino acid sequences that can be used to label proteins. The tag is fused to a recombinant protein using genetic engineering. In general protein tags are used for purification and detection via affinity chromatography, pull-down assays, western blot, immunohistochemistry, fluorescence microscopy, or in live imaging. Due to their structure, tags have different properties and, based on this, different areas of application. Tags are usually engineered onto either the N- or C- terminus in order to minimize tertiary structure disruptions that may alter protein function. Often a protease cleavage site can be inserted between the recombinant protein and the tag to proteolytically dissociate the tag after purification.

What are Affinity Tags?

Affinity tags are attached to proteins to faciliate purification from their biological source using an affinity technique. These include Strep tag and AVI Tag for example which both have a high affinity for biotin. The binding of biotin to streptavidin/avidin is one of the strongest non-covalent interactions known in nature.

The poly(His) tag is a widely used protein tag, which binds to divalent metal ions immobilized on solid phase matrices. His tagged proteins have increased polarity. In alkaline conditions, the histidine residues chelate the metal ion and bind to the carrier.

Fusion Protein Tags

Some affinity tags like maltose binding protein (MBP) or glutathione-S-transferase (GST) are not short AA sequences but large fusion proteins. Beside purification these two have a dual role as a solubilization agent. They ensure proper folding of proteins expressed in chaperone-deficient species such as E. coli and prevent precipitation.

What are Epitope Tags?

Epitope tags are artificial epitopes, which are cloned in frame with the coding sequence of the protein of interest. Epitope tags are typically composed of amino acid squenences of 10-15 amino acids in length. Due to their relatively small size, they have virtually no effect on the structure of the resulting fusion protein. Most epitope tags are derived from viral genes, which explains their high immunoreactivity. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. Examples are Myc-tag, HA-tag, Spot-tag, or V5-tag.

The DYKDDDDK tag, also known as FLAG® tag is one of the most specific epitope tags artificially created. The FLAG tag's structure was optimized for compatibility with proteins it is attached to. Its hydrophilic nature minimizes effects on function, secretion, or transport of the fusion protein.

Protein Tags FAQ - More Insight and Background Information on Protein Tags

What are Protein Tags?

Protein tags are short amino acid sequences that are added to a protein of interest to facilitate its detection, purification, localization, and/or manipulation in various experimental settings.

What are Protein Tags being Used for?

Protein tags facilitate the detection, purification or localization of a protein of interest in experimental settings.

What Types of Proteins Tags are There?

Different types of tags can be utilized with your protein of interest. Fluorescent tags like GFP or RFP; affinity tags like AVI or HIS tag. Affinity tags with larger proteins, eg MBP, are also called fusion protein tags. Epitope tags are artificial epitopes, which are cloned in frame with the coding sequence of the protein of interest.

What are Protein Tags being Used for?

Protein tags facilitate the detection, purification or localization of a protein of interest in experimental settings.

What are Fluorescent Protein Tags Being Used for?

Main advantage of fluorescence tags like GFP, RFP and their derivates is the ability to give visual readout on a protein. They can be utilized in reporter assays as well as in fluorescence microscopy to monitor correct folding or expression over time.

What are Fusion Protein Tags?

Some affinity tags like maltose binding protein (MBP) or glutathione-S-transferase (GST) are not short AA sequences but large fusion proteins. Beside purification these two have a dual role as a solubilization agent. They ensure proper folding of proteins expressed in chaperone-deficient species such as E. coli and prevent precipitation.

How does epitope tagging work?

To add an epitope tag to a protein, a DNA sequence encoding the tag is fused to the gene encoding the protein of interest. The resulting DNA construct is then used to express the fusion protein in cells or organisms. The tag is typically added at the N- or C- terminus of the protein, allowing it to be located on the surface of the protein and easily accessible for antibody binding.

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