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Antibodies to Peptides and Fusion Tags

By: Leigh Gaskill, BioFiles 2008, 3.6, 14.

BioFiles 2008, 3.6, 14.

Epitope Tag Antibody Detection

Unlike DNA and RNA, which can be targeted with complementary oligonucleotides, protein detection and purification usually relies upon specific antibodies. Although many are available commercially, not all proteins are catered for, especially if they have novel or unknown sequences.

Raising polyclonal antibodies is time-consuming and expensive; raising monoclonal even more so. Epitope tags (also known as fusion tags or affinity tags) offer a convenient solution to this problem by acting as universal epitopes for detection and purification without disturbing the structure of the protein they are fused.

The ideal tag for detection would be large and hydrophilic with a strong antibody recognition site positioned in an exposed region of the protein. However, large tags can interfere with protein structure, and exposed regions are often functionally active, so choosing a tag is not trivial.

Larger tags are often easier to detect because they are sterically more accessible to antibodies. Even in the presence of SDS, smaller tags can sometimes be folded inside the host protein and evade detection. Using a tag that incorporates charged residues helps overcome this because their hydrophilicity drives them to the surface and provides a strong motif for antibody recognition.

The FLAG® epitope is a practical example of how size and charge phenomena can exploited; it is only eight amino acids long but all residues are charged (DYKDDDDK), so detection is very sensitive. This sensitivity is enhanced 10-fold by tripling the size of the epitope to the 3xFLAG tandem repeat (DYKDHDGDYKDHDIDYKDDDK).

Table 1 Overview of Commonly Used Epitope Tags.
Epitope tags are classified by application into Detection and Purification tags. In general, detection tags are more sensitive than purification tags. However, purification tags have practical advantages.

Immunofluorescence COS 7 cells transfected with pFLAG-CMV2- BAP plasmid stained with ANTI-FLAG M2 FITC Conjugate (Cat No. F4049).

Immunoblotting Extract of 293-T cells transfected with pCDNA 3.1 (-)/Myc-His/LacZ plasmid (Cat No. C3215). (Lanes 1, 2) and extract of 293-T untransfected (Lane 3) were separated on SDS-PAGE and blotted with Monoclonal Anti-c-Myc-Biotin (Cat No. B7554). The antibody was developed with ExtrAvidin®-Peroxidase (Cat No. E2886). and chemiluminescent substrate. (Lanes 1, 3) Antibody concentration 0.05 mg/mL; (Lane 2) Without primary antibody + ExtrAvidin -Peroxidase dilution: 1:20,000.

Immunofluorescence Immunofluorescence Cytoplasmic localization of V5-HIS-LacZ fusion protein in transfected HEK-293T cells, stained with 2 μg/mL Monoclonal Anti-V5-Cy3 conjugate (Clone V5-10) (Cat No. V4014).

Immunoblotting Decreasing amount of recombinant GFP were loaded onto lanes of a mini gel separated on SDS-PAGE, blotted with Rabbit Anti-GFP (N-Terminal) (Cat No. G1544) and developed using Goat Anti-Rabbit IgG-Peroxidase and a chemiluminescent substrate. Protein loaded per lane ranged from 3−200 ng.

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