Insertion of tags
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Insertion of tags

Seattle Genova provides fast and efficient molecular cloning services to our customers. Our experienced scientists can add any protein tags to your existing plasmids. Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. At present, there is a wide variety of protein tags that can be used to suit your purpose. These include affinity tags, epitope tags and fluorescent tags.

Affinity Tags
Affinity tagged proteins can be purified by using a specific affinity resin while detection can be facilitated by using commercially available antibodies. Some of the most commonly used affinity tags include the following:

· Glutathione-S transferase (GST)

GST has a strong binding affinity for GSH, beads coated with the compound can be added to the mixture to isolate your protein of interest from the rest of the solution.

· Poly-Histidine tag (His)

It comprises of 6-8 histidine residues. His-tagged proteins are purified using immobilized nickel, cobalt or zinc ions, and eluted using EDTA or imidazole.

· Calmodulin Binding Protein (CBP)

The tag binds to a calmodulin resin and the proteins can be eluted with a neutral buffer containing low concentrations of EGTA, a calcium chelator.

· Maltose-binding protein (MBP)

These tags bind to amylase agarose and are commonly used to increase the solubility of fusion proteins.

Epitope Tags
Due to their relatively small size, they have extremely little or no effect on the structure of the resulting fusion protein. Epitope tags are ideal for several downstream applications which include western blotting, co-immunoprecipitation and immunofluorescence experiments. Some of the most popular epitope tags include the following:

· Myc tag

This tag is a short peptide sequence (EQKLISEEDL ) derived from the c-myc gene product and recognized by numerous commercial antibodies.

· Human influenza hemagglutinin (HA) tag

The HA tag is a peptide sequence (YPYDVPDYA) derived from the surface glycoprotein that facilitates the ability of the influenza virus to infect its host and is recognized by numerous commercial antibodies.

· FLAG tag

The FLAG tag is a popular short peptide tag (DYKDDDDK) used in recombinant DNA technology. The tag is more hydrophilic as compared to other tags in its class so they do not denature or inactivate the proteins to which they are attached.

Fluorescent Tags

· Green fluorescent proteins

Green fluorescent protein (GFP) is a protein in the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light.

· mCherry

mCherry is probably the most commonly used RFP variant. It is a monomeric red fluorescent protein with broad applicability as a fusion protein in various cell types. Like other mFruit RFPs, mCherry is derived from the dsRed variant mRFP1 via directed evolution by Roger Tsien’s lab. Compared to other mFruits, mCherry has the highest photostability, fastest maturation rate, and excellent pH resistance. It has, however, a lower quantum yield than mRFP1.

Insertion of tags

Seattle Genova provides fast and efficient molecular cloning services to our customers. Our experienced scientists can add any protein tags to your existing plasmids. Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. At present, there is a wide variety of protein tags that can be used to suit your purpose. These include affinity tags, epitope tags and fluorescent tags.

Affinity Tags
Affinity tagged proteins can be purified by using a specific affinity resin while detection can be facilitated by using commercially available antibodies. Some of the most commonly used affinity tags include the following:

· Glutathione-S transferase (GST)

GST has a strong binding affinity for GSH, beads coated with the compound can be added to the mixture to isolate your protein of interest from the rest of the solution.

· Poly-Histidine tag (His)

It comprises of 6-8 histidine residues. His-tagged proteins are purified using immobilized nickel, cobalt or zinc ions, and eluted using EDTA or imidazole.

· Calmodulin Binding Protein (CBP)

The tag binds to a calmodulin resin and the proteins can be eluted with a neutral buffer containing low concentrations of EGTA, a calcium chelator.

· Maltose-binding protein (MBP)

These tags bind to amylase agarose and are commonly used to increase the solubility of fusion proteins.

Epitope Tags
Due to their relatively small size, they have extremely little or no effect on the structure of the resulting fusion protein. Epitope tags are ideal for several downstream applications which include western blotting, co-immunoprecipitation and immunofluorescence experiments. Some of the most popular epitope tags include the following:

· Myc tag

This tag is a short peptide sequence (EQKLISEEDL ) derived from the c-myc gene product and recognized by numerous commercial antibodies.

· Human influenza hemagglutinin (HA) tag

The HA tag is a peptide sequence (YPYDVPDYA) derived from the surface glycoprotein that facilitates the ability of the influenza virus to infect its host and is recognized by numerous commercial antibodies.

· FLAG tag

The FLAG tag is a popular short peptide tag (DYKDDDDK) used in recombinant DNA technology. The tag is more hydrophilic as compared to other tags in its class so they do not denature or inactivate the proteins to which they are attached.

Fluorescent Tags

· Green fluorescent proteins

Green fluorescent protein (GFP) is a protein in the jellyfish Aequorea Victoria that exhibits green fluorescence when exposed to light.

· mCherry

mCherry is probably the most commonly used RFP variant. It is a monomeric red fluorescent protein with broad applicability as a fusion protein in various cell types. Like other mFruit RFPs, mCherry is derived from the dsRed variant mRFP1 via directed evolution by Roger Tsien’s lab. Compared to other mFruits, mCherry has the highest photostability, fastest maturation rate, and excellent pH resistance. It has, however, a lower quantum yield than mRFP1.

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