Nano-Tag9 Monoclonal Antibody

What is Nano-Tag9 and how does it function in protein research?

Nano-Tag9 is a short peptide sequence (MDVEAWLGAR) that functions as an epitope tag in recombinant protein research. It belongs to a family of streptavidin-binding peptides that possess nanomolar affinity for streptavidin, hence the name "Nano-tag" . Epitope tags like Nano-Tag9 are short peptide sequences that can be genetically fused to proteins of interest, enabling their detection and purification without the need to generate specific antibodies against the target protein itself.

The Nano-Tag9 functions through two main mechanisms in protein research. First, it can be used for protein purification through its strong interaction with streptavidin, allowing researchers to isolate tagged proteins using streptavidin-coated resins or beads. With a dissociation constant of 17 nM, Nano-Tag9 binds streptavidin tightly enough for efficient purification while still allowing elution under mild conditions .

Second, Nano-Tag9 serves as an epitope recognized by specific monoclonal antibodies, enabling the detection of tagged proteins in various experimental techniques, most commonly Western blotting . This dual functionality makes Nano-Tag9 a versatile tool in protein expression studies, allowing researchers to both purify and detect proteins of interest using a single tag.

How does Nano-Tag9 compare to other common epitope tags?

When comparing Nano-Tag9 to other commonly used epitope tags, several factors must be considered including size, binding affinity, versatility, and potential interference with protein function. Below is a comparative analysis of Nano-Tag9 versus other popular epitope tags used in protein research:

The key advantage of Nano-Tag9 is its relatively small size combined with its high affinity for streptavidin (Kd = 17 nM), which is significantly stronger than many other affinity tags like His6 or Strep-tag II . This enables more stringent washing conditions during purification while maintaining binding. Additionally, unlike some traditional epitope tags, Nano-Tag9 offers dual functionality for both detection (via monoclonal antibodies) and purification (via streptavidin binding).

What are the optimal experimental conditions for using Nano-Tag9 Monoclonal Antibody in Western blotting?

Based on the available information from multiple antibody suppliers, here are the optimal experimental conditions for using Nano-Tag9 Monoclonal Antibody in Western blotting:

Dilution Recommendations:

The recommended working dilutions for Nano-Tag9 Monoclonal Antibody in Western blotting vary somewhat between suppliers but generally fall within these ranges:

• 1:1000 to 1:10000 (Sunlong Biotech)

• 1:2000 to 1:5000 (Abbkine)

• 1:300 to 1:5000 (Bioss)

For optimal results, researchers should perform a dilution series to determine the ideal concentration for their specific experimental conditions and detection system.

Sample Preparation:

• Load appropriate amounts of protein containing the Nano-Tag9 epitope (typically 2-4 μg of recombinant protein as shown in the Western blot examples)

• Include positive controls such as recombinant Nano-Tag9 fusion proteins

• Include negative controls (non-tagged proteins) to verify antibody specificity

Primary Antibody Incubation:

• Dilute antibody in blocking buffer according to the recommended dilutions

• Incubate membranes with diluted primary antibody for 1-2 hours at room temperature or overnight at 4°C

• Wash 3-5 times with TBS-T after primary antibody incubation

Secondary Antibody Selection:

Since most Nano-Tag9 monoclonal antibodies are mouse IgG or IgG1 isotype , use:

• Anti-mouse IgG secondary antibody conjugated to HRP, fluorescent dye, or other detection system

• Some suppliers offer direct HRP-conjugated Nano-Tag9 antibodies (e.g., Bioss bsm-33215M-HRP) which eliminate the need for secondary antibody incubation

Western blot images provided by suppliers show clean detection of Nano-Tag9 fusion proteins with minimal background, indicating high specificity of these antibodies when used under optimal conditions.

How should Nano-Tag9 Monoclonal Antibody be stored and handled for maximum efficacy?

Proper storage and handling of Nano-Tag9 Monoclonal Antibody is crucial for maintaining its activity and specificity over time. Based on the information provided by multiple suppliers, here are the recommended storage and handling guidelines:

Storage Temperature:

• Long-term storage: -20°C is unanimously recommended by all suppliers

• Working aliquots: 4°C for short-term use (up to a few weeks)

Storage Buffer Composition:

The antibodies are typically provided in a stabilized buffer containing:

• PBS or TBS (pH 7.4) as the base buffer

• 50% Glycerol as a cryoprotectant to prevent freeze damage

• BSA (0.5-1%) as a stabilizer and to prevent non-specific binding

• Preservatives such as 0.02-0.03% sodium azide or 0.03% Proclin300 to prevent microbial growth

Aliquoting Recommendations:

• Upon receiving the antibody, it's advisable to prepare smaller working aliquots to avoid repeated freeze-thaw cycles

• Use sterile tubes and sterile technique when preparing aliquots

• Typical aliquot volumes of 10-20 μL are practical for most experimental needs

Freeze-Thaw Considerations:

• All suppliers emphasize avoiding repeated freeze-thaw cycles as they can lead to protein denaturation and loss of antibody activity

• After thawing an aliquot, centrifuge the vial briefly before opening to collect all liquid at the bottom of the tube

Shelf Life:

• Typical shelf life when stored properly at -20°C is approximately one year from date of receipt

• Activity may gradually decrease over time even with proper storage

By following these storage and handling recommendations, researchers can maximize the lifespan and performance of their Nano-Tag9 Monoclonal Antibody, ensuring consistent and reliable results in their experiments.

What is the mechanism of interaction between Nano-Tag9 and streptavidin?

The interaction between Nano-Tag9 (MDVEAWLGAR) and streptavidin involves specific molecular recognition that results in the nanomolar affinity (dissociation constant of 17 nM) observed between these molecules . While the specific crystal structure of the Nano-Tag9/streptavidin complex is not detailed in the provided search results, we can infer the mechanism based on known properties of streptavidin-peptide interactions.

Streptavidin is a tetrameric protein with four binding pockets that typically interact with biotin with exceptionally high affinity. Nano-Tag9 was designed as an alternative binding peptide that occupies the same or nearby binding pocket, albeit with lower affinity than biotin.

The nanomolar binding affinity of Nano-Tag9 to streptavidin (17 nM) suggests that the interaction involves multiple non-covalent forces including:

• Hydrogen bonding between amino acid side chains of Nano-Tag9 and complementary residues in the streptavidin binding pocket

• Hydrophobic interactions, potentially involving the tryptophan (W) and alanine (A) residues in Nano-Tag9

• Van der Waals forces between closely positioned atoms

• Possible electrostatic interactions involving charged residues like aspartic acid (D) and arginine (R) in the peptide

The higher binding affinity of Nano-Tag15 (4 nM) compared to Nano-Tag9 (17 nM) suggests that the additional 6 amino acids (VPLVET) in Nano-Tag15 provide further stabilizing interactions with streptavidin . This difference in affinity provides researchers with options depending on their specific purification or detection needs - Nano-Tag9 might be preferred when milder elution conditions are desired, while Nano-Tag15 might be chosen when more stringent washing steps are needed.

How can Nano-Tag9 be used in multi-protein complex purification strategies?

Nano-Tag9's nanomolar affinity for streptavidin (Kd = 17 nM) makes it an excellent candidate for multi-protein complex purification strategies. This application leverages both the tag's strong binding affinity and the availability of specific monoclonal antibodies against the tag. Here's a methodological approach to using Nano-Tag9 in complex purification:

Tandem Affinity Purification (TAP) Strategy:

Nano-Tag9 can be incorporated into a TAP approach, where two or more affinity tags are used sequentially to achieve higher purity of protein complexes. A typical Nano-Tag9-based TAP strategy could involve:

• Design of fusion constructs:

  • Clone the Nano-Tag9 sequence (encoding MDVEAWLGAR) into an expression vector

  • Fuse it to one component of your protein complex of interest

  • Optionally, include a protease cleavage site between the tag and protein for tag removal

  • Consider position effects: N-terminal vs. C-terminal tagging may affect complex formation differently

• Primary capture using streptavidin:

  • Lyse cells expressing the Nano-Tag9-fused protein under conditions that preserve protein-protein interactions

  • Incubate lysate with streptavidin-coated beads or resin

  • Wash extensively to remove non-specifically bound proteins

  • Elute using biotin competition or other elution methods

• Secondary purification options:

  • Option A: If using dual tags, proceed with the second affinity step

  • Option B: Use Nano-Tag9 Monoclonal Antibody for immunoprecipitation

  • Option C: Use size exclusion chromatography to separate complexes by size

Methodological Considerations:

For optimal results when purifying multi-protein complexes with Nano-Tag9:

• Buffer optimization:

  • Test different lysis and binding buffers to maintain complex integrity

  • Consider adding stabilizers like glycerol (5-10%)

  • Include protease inhibitors to prevent degradation

  • Test different salt concentrations to optimize specificity vs. complex stability

• Elution strategies for streptavidin binding:

  • Competitive elution with biotin or biotin derivatives

  • For gentler elution, consider using Nano-Tag9 synthetic peptide as a competitor

  • If using TEV protease cleavage site, enzymatic elution may provide higher specificity

By strategically employing Nano-Tag9 in protein complex purification workflows, researchers can achieve highly specific isolation of native protein complexes while minimizing the risk of tag-induced artifacts in complex composition or function.

How does the binding affinity of Nano-Tag9 vs Nano-Tag15 affect experimental design?

The difference in binding affinity between Nano-Tag9 (Kd = 17 nM) and Nano-Tag15 (Kd = 4 nM) to streptavidin creates distinct considerations for experimental design. This approximately 4-fold difference in affinity significantly impacts purification strategies, detection sensitivity, and potential applications of these tags.

Implications for Protein Purification:

• Washing Stringency:

  • Nano-Tag15 (Kd = 4 nM): Tolerates more stringent washing conditions, reducing non-specific background

  • Nano-Tag9 (Kd = 17 nM): Requires more careful optimization of washing buffers to maintain specific binding

  Methodological approach: For Nano-Tag9 purifications, consider using higher concentrations of non-ionic detergents (e.g., 0.1-0.5% Triton X-100) rather than high salt to reduce non-specific binding while maintaining tag-streptavidin interaction.

• Elution Efficiency:

  • Nano-Tag15: More difficult to elute from streptavidin, may require harsher conditions

  • Nano-Tag9: Easier to elute using milder conditions, potentially preserving protein activity

  Methodological approach: For Nano-Tag9, competitive elution with 2-5 mM biotin may be sufficient, while Nano-Tag15 might require biotin with additional denaturing agents or higher concentrations.

Impact on Experimental Applications:

Structural Considerations:

The additional 6 amino acids in Nano-Tag15 (VPLVET) compared to Nano-Tag9 may also impact:

• Protein Folding and Function:

  • The longer tag may potentially interfere more with protein folding

  • Terminal flexibility may differ between the two tags

  Methodological approach: If protein function is affected with Nano-Tag15, try Nano-Tag9 or alter the linker sequence between tag and protein.

By carefully considering these differences in binding affinity and their experimental implications, researchers can select the optimal Nano-Tag variant for their specific application, potentially maximizing both experimental efficiency and data quality.

What are the best strategies for troubleshooting non-specific binding with Nano-Tag9 Monoclonal Antibody?

Non-specific binding can be a significant challenge when using monoclonal antibodies, including those against Nano-Tag9. Based on the properties of Nano-Tag9 Monoclonal Antibody and general immunological principles, here are comprehensive troubleshooting strategies organized by problem type:

High Background in Western Blotting

• Optimize Antibody Dilution:

  • Perform a dilution series (e.g., 1:1000, 1:2000, 1:5000, 1:10000) to determine optimal concentration

  • The recommended dilution range for Nano-Tag9 antibodies varies from 1:300 to 1:10000 depending on the supplier

  • Higher dilutions generally reduce background but may also reduce specific signal

• Improve Blocking Efficiency:

  • Test different blocking agents: 5% non-fat milk vs. 3-5% BSA vs. commercial blocking buffers

  • Extend blocking time to 2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.3% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Optimize Washing Procedure:

  • Increase wash duration (5-10 minutes per wash)

  • Increase number of washes (5-6 times)

  • Use higher concentration of Tween-20 in wash buffer (0.1-0.2%)

Cross-Reactivity Issues

• Validate Antibody Specificity:

  • Run parallel blots with positive control (Nano-Tag9-tagged protein) and negative control (untagged protein)

  • Use competitive blocking with synthetic Nano-Tag9 peptide (MDVEAWLGAR) to confirm specificity

  • The Nano-Tag9 sequence is unique enough that cross-reactivity should be minimal

• Pre-adsorption Strategy:

  • Pre-incubate diluted antibody with lysate from cells not expressing Nano-Tag9-tagged proteins

  • Centrifuge and use the supernatant for immunodetection

  • This removes antibodies that bind to endogenous proteins

• Alternative Clone Selection:

  • Different suppliers offer different monoclonal clones (e.g., clone 6B7 , clone 4C1 , clone 11T3 )

  • Test multiple clones as they may have different cross-reactivity profiles

  • Some clones may perform better in certain applications or with certain sample types

Sample-Related Issues

• Optimize Protein Loading:

  • Reduce total protein amount if background is high

  • For recombinant Nano-Tag9 fusion proteins, 2-4 μg appears optimal based on supplier data

  • Ensure even loading across wells using a loading control

• Sample Buffer Composition:

  • Ensure complete protein denaturation (boil samples in SDS sample buffer)

  • Add reducing agent (DTT or β-mercaptoethanol) to break disulfide bonds

  • Consider adding urea (up to 8M) for highly hydrophobic proteins

By systematically applying these troubleshooting strategies, researchers can optimize the performance of Nano-Tag9 Monoclonal Antibody for their specific experimental conditions and sample types, maximizing specificity while minimizing background and cross-reactivity issues.

How can Nano-Tag9 be integrated into CRISPR-Cas9 gene editing workflows?

Integrating Nano-Tag9 into CRISPR-Cas9 gene editing workflows offers powerful opportunities for endogenous protein tagging and functional genomics studies. This advanced application combines the precision of CRISPR-Cas9 with the advantages of the Nano-Tag9 system. Here's a comprehensive methodological approach:

Endogenous Protein Tagging Strategy

• Design Considerations for Knock-in:

  • Select appropriate genomic location for tag insertion (N-terminus, C-terminus, or internal)

  • For C-terminal tagging, design guide RNA targeting near the stop codon

  • For N-terminal tagging, target near the start codon

  • Consider protein domain structure to avoid disrupting functional regions

• Delivery Methods:

  • Transfection of Cas9/gRNA expression plasmids with donor template

  • Ribonucleoprotein (RNP) delivery with synthetic donor template

  • Viral delivery systems for difficult-to-transfect cells

  • For highest efficiency, consider using Cas9 nickase with paired gRNAs to reduce off-target effects

Validation and Detection of Nano-Tag9 Knock-in

• Initial Screening:

  • Design PCR primers spanning the integration junction

  • Perform genomic PCR to identify potential positive clones

  • Confirm proper integration by Sanger sequencing

• Protein Expression Validation:

  • Western blot using Nano-Tag9 Monoclonal Antibody (1:1000-1:5000 dilution)

  • Immunoprecipitation with streptavidin beads to confirm streptavidin binding functionality

  • Immunofluorescence to verify expected subcellular localization

• Functional Validation:

  • Compare protein activity/function before and after tagging

  • Assess if tag affects protein-protein interactions

  • Verify that cellular phenotype is unchanged in tagged cell lines

Advanced Applications of Nano-Tag9 in CRISPR-Edited Cells

• Proteomics Analysis:

  • Use streptavidin-based affinity purification to isolate endogenous protein complexes

  • Perform mass spectrometry to identify interaction partners

  • Compare interactome data under different cellular conditions

• ChIP-seq for DNA-Binding Proteins:

  • Perform chromatin immunoprecipitation using Nano-Tag9 Monoclonal Antibody

  • Sequence bound DNA to map genomic binding sites

  • The small size of Nano-Tag9 minimizes interference with DNA binding

By following this methodological framework, researchers can effectively integrate Nano-Tag9 into CRISPR-Cas9 gene editing workflows, enabling precise endogenous protein tagging for a wide range of advanced protein analysis applications while minimizing potential artifacts associated with overexpression systems.

What are the considerations for using Nano-Tag9 in microRNA detection systems?

While the primary applications of Nano-Tag9 focus on protein studies, researchers have explored adapting similar tag systems for nucleic acid detection approaches, including microRNA profiling. Based on information in search result which discusses a universal tag approach for microRNA detection, we can extrapolate methodological considerations for implementing Nano-Tag9 in similar systems:

Oligonucleotide Tag Design Considerations

Research on universal tag systems for microRNA detection has demonstrated that tag length is critical for optimal performance. As noted in the cited study, they tested various oligomer lengths and found that an 8-mer universal tag provided optimal specificity and hybridization properties . This finding may be relevant when considering Nano-Tag9-inspired approaches for nucleic acid detection.

The study noted: "Too short a UT, the duplex could be unlikely to form even if the targets had hybridized to the probes to provide the stacking sites; too long a UT, it could maintain a stable hybridization to the probe without the target miRNAs" . This principle would likely apply to any tag-based detection system.

Potential Methodological Approach for Nano-Tag9 in RNA Detection

• Design of Nano-Tag9-Based Nucleic Acid Probes:

  • Create DNA/RNA oligonucleotides containing the Nano-Tag9 sequence

  • Design chimeric oligonucleotides with a target-specific portion and the tag sequence

  • Consider optimal hybridization temperature based on the sequence composition

• Signal Amplification Strategy:

  • Use streptavidin-conjugated detection molecules to bind to Nano-Tag9 sequences

  • Employ Nano-Tag9 Monoclonal Antibody as a detection reagent for bound oligonucleotides

  • Consider multiplexed detection using Nano-Tag9 in combination with other epitope tags

• Optimization Parameters:

  • Test hybridization stringency conditions to maximize signal-to-noise ratio

  • Determine optimal probe/tag hybrid lengths for specific applications

  • Evaluate detection sensitivity limits compared to standard methods

The research on universal tags for microRNA detection demonstrates that careful optimization of tag length and hybridization conditions is essential for developing effective nucleic acid detection systems . Researchers interested in adapting Nano-Tag9 for such applications would need to conduct similar optimization studies specific to this tag sequence.