V5-Tag Monoclonal Antibody

What is the V5 epitope tag and what is its origin?

The V5 epitope tag, originally known as the Pk tag, was developed in the early 1990s by Professor R. Randall's group at the University of St. Andrews. It is derived from a small epitope present on the P/V proteins of the paramyxovirus of simian virus 5 (SV5) family . The tag was initially developed as part of research focused on new vaccine production targeted at HIV and SIV, where the team needed a method to capture and immobilize SIV proteins in solid matrix-antibody-antigen (SMAA) complexes . The Randall laboratory identified this linear epitope and produced a highly specific monoclonal antibody against it, which they demonstrated could be used for detection and purification of recombinant proteins .

What is the amino acid sequence of the V5 tag and how is it typically used?

The complete V5 tag consists of 14 amino acids with the sequence GKPIPNPLLGLDST, although a shorter 9-amino acid epitope (IPNPLLGLD, which is underlined in the full sequence) has also been successfully used in research applications . The tag is typically added to either the N-terminus or C-terminus of recombinant proteins through molecular cloning techniques . This addition enables researchers to detect and purify the protein of interest without requiring a protein-specific antibody, which is particularly valuable when studying novel proteins for which specific antibodies may not be available .

What are the primary applications of V5-Tag Monoclonal Antibodies in research?

V5-Tag Monoclonal Antibodies have been validated for multiple research applications:

• Western Blotting: Detection of V5-tagged proteins in cell or tissue lysates, typically at dilutions of 1:100 to 1:50000 depending on the specific antibody .

• Immunoprecipitation: Purification of V5-tagged proteins from complex biological samples, often used at dilutions of 1:2000 to 1:20000 .

• Immunofluorescence: Visualization of the subcellular localization of V5-tagged proteins within cells .

• Flow Cytometry: Quantitative analysis of V5-tagged protein expression in cell populations .

• ELISA: Quantification of V5-tagged proteins in solution .

• Simple Western™: Automated capillary-based immunoassay for protein detection .

These applications have been demonstrated across various experimental systems, including different cell types and diverse recombinant proteins with both N-terminal and C-terminal tags .

How does the positioning of the V5 tag affect protein detection and function?

When designing V5-tagged constructs, researchers should consider:

• The structural features of the target protein, including known functional domains

• Whether the N- or C-terminus participates in crucial protein-protein interactions

• The presence of signal sequences or localization signals that might be disrupted by tag placement

In some cases, tag positioning can disrupt protein structure and function. For example, in the development of a diagnostic reagent for West Nile virus, researchers found that using a polyhistidine tag "disrupted" the prM antigen structure, whereas the V5-tag "allowed formation of the authentic antigenic structure" while still enabling proper presentation of the tag itself . This highlights the importance of careful consideration when selecting both the tag type and its position.

What are the optimal conditions for using V5-Tag Monoclonal Antibodies in Western blotting?

For optimal Western blotting results with V5-Tag Monoclonal Antibodies, the following conditions have been experimentally validated:

• Sample preparation: Lysates of cells expressing V5-tagged proteins should be prepared under reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 1) .

• Loading amount: Typically 25 μg of total protein per lane is sufficient for detection of moderately expressed V5-tagged proteins .

• Membrane type: PVDF membranes have been successfully used for transfer of V5-tagged proteins .

• Antibody dilution: The optimal dilution range varies by antibody clone and manufacturer:

  • Clone SV5-Pk4: 1:100 to 1:500

  • Other V5 antibodies: 1:5000 to 1:50000

• Detection system: HRP-conjugated secondary antibodies followed by enhanced chemiluminescence provide sensitive detection of V5-tagged proteins .

A representative example showed detection of V5-tagged proteins at approximately 110 kDa (CBL), 34 kDa (FSNC4), 75 kDa (LAMP2B), and 65 kDa (GALNT2) in transfected HEK293 cells . This demonstrates the versatility of V5-Tag Monoclonal Antibodies in detecting proteins across a wide molecular weight range.

How can V5-Tag Monoclonal Antibodies be used effectively in immunofluorescence studies?

For successful immunofluorescence detection of V5-tagged proteins, the following protocol has been validated:

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes .

• Permeabilization: Permeabilize with 0.25% Triton X-100 for 10 minutes .

• Blocking: Block with 5% BSA for 1 hour at room temperature .

• Primary antibody: Incubate with V5-Tag Monoclonal Antibody diluted in 1% BSA (typically 1:500 dilution) for 3 hours at room temperature .

• Secondary antibody: Label with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488-conjugated anti-mouse IgG) at 1:400 dilution for 30 minutes .

• Nuclear counterstain: Use DAPI or similar DNA stain for nuclear visualization .

• Additional counterstain options: F-actin can be visualized with fluorescently labeled phalloidin to provide cellular context .

This approach has successfully demonstrated cytoplasmic localization of V5-His/LacZ in transfected HEK-293 cells, with appropriate controls using untransfected cells to confirm specificity .

What are common issues in V5-tagged protein detection and how can they be resolved?

When working with V5-tagged proteins, researchers may encounter several challenges that can be addressed through specific troubleshooting approaches:

• High background signal:

  • Problem: Non-specific binding of V5 antibody to endogenous proteins

  • Solution: Use newer monoclonal antibodies such as those described in 1999, which "gave less background immunofluorescence than the original" . Optimize blocking conditions using 5% BSA and include appropriate detergents in wash buffers.

• Poor detection sensitivity:

  • Problem: Insufficient expression of V5-tagged protein or epitope masking

  • Solution: Verify expression levels using multiple detection methods. For example, combine Western blot analysis with flow cytometry as demonstrated in validation studies where both techniques confirmed expression of V5-tagged proteins in transfected HEK293 cells .

• Tag interference with protein function:

  • Problem: V5 tag disrupts protein folding or activity

  • Solution: Compare N-terminal versus C-terminal tagging, or consider using the shorter 9-amino acid version of the tag which may cause less interference while still enabling detection .

• Sample degradation:

  • Problem: Proteolytic degradation of tagged proteins

  • Solution: Include protease inhibitors in lysis buffers and handle samples at appropriate temperatures to maintain protein integrity.

How can V5-Tag Monoclonal Antibodies be used in flow cytometry applications?

Flow cytometry using V5-Tag Monoclonal Antibodies enables quantitative analysis of V5-tagged protein expression at the single-cell level. The following protocol has been validated for this application:

• Cell fixation: Fix cells with 70% ethanol for 10 minutes or use 4% paraformaldehyde .

• Permeabilization: Permeabilize with 0.25% Triton X-100 for 20 minutes to allow antibody access to intracellular targets .

• Blocking: Block with 5% BSA for 1 hour at room temperature to reduce non-specific binding .

• Antibody staining: Incubate cells with V5-Tag Monoclonal Antibody, followed by fluorophore-conjugated secondary antibody .

• Controls: Include isotype control antibodies (such as catalog # AB-105-C) to establish gating parameters .

This approach has successfully demonstrated detection of V5-tagged proteins in transfected cell populations, with clear discrimination between positive and negative cells . For example, flow cytometry analysis of HEK293 cells transfected with V5-tagged proteins showed strong positive staining with V5-Tag Monoclonal Antibody compared to the isotype control .

What are the advantages of V5 tag compared to other epitope tags in specific research contexts?

The V5 tag offers several distinct advantages over alternative tagging systems in certain research contexts:

• Structural preservation: The V5 tag has been shown to maintain authentic protein structure in cases where other tags caused disruption. For example, in West Nile virus diagnostic reagent development, the V5 tag preserved the antigenic structure of the prM protein while a polyhistidine tag disrupted it .

• Versatility across protein types: V5-Tag Monoclonal Antibodies have successfully detected diverse proteins including transmembrane proteins (LAMP2B), glycosylated proteins (GALNT2), and cytosolic proteins (CBL) .

• Compatibility with multiple detection methods: The V5 tag system has been validated across the full spectrum of protein analysis techniques including Western blotting, immunoprecipitation, immunofluorescence, and flow cytometry .

• Size options: Researchers can choose between the full 14-amino acid tag or the shorter 9-amino acid version depending on experimental requirements .

• Established specificity: The V5-Tag Monoclonal Antibodies have been extensively characterized for specificity, with minimal cross-reactivity to endogenous proteins in mammalian cells .

These advantages make the V5 tag particularly suitable for applications requiring preserved protein conformation and function, such as structural studies, protein-protein interaction analyses, and development of diagnostic reagents.

How have V5-Tag Monoclonal Antibodies contributed to virus research?

V5-Tag Monoclonal Antibodies have made significant contributions to virus research, particularly in studies of viral-host protein interactions and diagnostic reagent development:

• SIV/HIV vaccine development: The V5 tag system was originally developed as part of efforts to produce vaccines against SIV and HIV. The Randall laboratory used the V5 tag to create solid matrix-antibody-antigen (SMAA) complexes containing SIV p27 protein for immunization studies .

• Rabies virus research: V5-Tag Monoclonal Antibodies have been used to study interactions between rabies virus phosphoprotein and host factors. Research by Li et al. (2016) used V5-tagged proteins to investigate how rabies virus phosphoprotein interacts with ribosomal protein L9, providing insights into virus replication mechanisms .

• West Nile virus diagnostics: The V5 tag played a crucial role in the development of diagnostic reagents for West Nile virus. Researchers created a V5-tagged version of the prM antigen that retained its authentic antigenic structure, enabling the development of a "useful diagnostic agent" for detecting antibodies against West Nile virus in serum samples .

• Paramyxovirus studies: Given that the V5 tag originates from paramyxovirus SV5, it has been particularly valuable in paramyxovirus research, enabling studies of viral protein function and interactions .

These applications demonstrate how V5-Tag Monoclonal Antibodies have facilitated advances in understanding virus biology and developing tools for virus detection and prevention.

What protein interaction studies have benefited from V5-tagged proteins?

V5-tagged proteins have been instrumental in elucidating protein-protein interactions across diverse biological contexts:

• Ubiquitin signaling pathway: Early studies at the University of St. Andrews by the Hay laboratory utilized V5-tagged proteins to investigate protein interactions within the ubiquitin signaling pathway . This research helped establish the broad utility of the V5-tag/antibody system for studying protein-protein interactions.

• Viral protein interactions: The Randall laboratory employed V5-tagged viral proteins to study protein-protein interactions in viruses, contributing to our understanding of viral replication mechanisms .

• Transmembrane and secreted proteins: V5-Tag Monoclonal Antibodies have been used to detect and study interactions involving transmembrane and secreted proteins, demonstrating the versatility of this tagging system across different protein classes .

• Co-immunoprecipitation studies: V5-Tag Monoclonal Antibodies have been validated for immunoprecipitation applications, enabling researchers to pull down V5-tagged proteins along with their interaction partners for subsequent analysis .

These studies highlight how V5-tagging approaches have advanced our understanding of complex protein interaction networks in both normal cellular processes and disease states.

What are the recommended storage conditions for V5-Tag Monoclonal Antibodies?

For optimal preservation of antibody activity and specificity, V5-Tag Monoclonal Antibodies should be stored according to the following guidelines:

• Storage temperature: Store at -20°C for long-term preservation .

• Formulation: Most commercial preparations are supplied as purified IgG in phosphate-buffered saline (PBS) with preservatives such as 0.09% sodium azide and stabilizers like 50% glycerol (pH 7.3) .

• Freeze-thaw cycles: Avoid repeated freezing and thawing as this may denature the antibody and reduce its activity .

• Frost-free freezers: Storage in frost-free freezers is not recommended as temperature fluctuations can compromise antibody stability .

• Aliquoting: Do not aliquot the antibody unless specifically recommended by the manufacturer .

• Shelf life: Most manufacturers provide a guarantee of 12 months from the date of dispatch when stored according to recommendations .

Following these storage guidelines will help ensure consistent antibody performance across experiments and maximize the useful life of V5-Tag Monoclonal Antibodies.

What are the characteristics of commonly used V5-Tag Monoclonal Antibody clones?

Several V5-Tag Monoclonal Antibody clones are commercially available, each with specific characteristics:

Most V5-Tag Monoclonal Antibodies are produced by immunizing mice (typically BALB/c strain) with recombinant peptides corresponding to the V5/Pk tag fused to carrier proteins or helper cell epitopes . The resulting antibodies are typically purified by affinity chromatography on Protein A from tissue culture supernatant .