FLAG PAT3G6AT antibody

What is the FLAG tag system and how does the PAT3G6AT antibody fit into it?

The FLAG tag is an artificial fusion tag consisting of eight amino acids (AspTyrLysAspAspAspAspLys, or DYKDDDDK) that includes an enterokinase-cleavage site. It was specifically designed for immunoaffinity chromatography, enabling protein elution under non-denaturing conditions . The PAT3G6AT antibody is a mouse monoclonal antibody developed through hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice that were immunized with a synthetic DYKDDDDKC-KLH peptide . This antibody belongs to the IgG class with a heavy chain and kappa light chain, and is purified from mouse ascitic fluids by protein-A affinity chromatography . PAT3G6AT specifically recognizes the FLAG epitope, making it valuable for detecting, localizing, and purifying FLAG-tagged recombinant proteins.

What are the primary applications of the FLAG tag system in molecular biology research?

The FLAG peptide system has multiple applications in contemporary molecular biology research, including:

• Western blotting for protein detection and quantification

• Immunocytochemistry for cellular localization studies

• Immunoprecipitation for protein-protein interaction analysis

• Flow cytometry for cell surface protein detection

• Protein purification via affinity chromatography

• Investigation of protein-protein interactions

• Analysis of cell ultrastructure

• Protein localization studies

The hydrophilic nature of the FLAG tag significantly enhances detection and purification efficiency of recombinant fusion proteins without substantially altering their structure or function .

How does the FLAG antibody detection system work compared to direct protein detection?

FLAG-based detection differs fundamentally from direct protein detection in that it targets a standardized epitope tag rather than variable protein-specific epitopes. This approach offers several methodological advantages:

• Standardization: The FLAG system uses a universal tag recognized by well-characterized antibodies, enabling consistent detection protocols across different target proteins .

• Circumvention of antibody limitations: When quality antibodies against specific proteins are unavailable, the FLAG system provides an alternative detection method by recognizing the tag rather than the protein itself .

• Expression control: In knock-in mice with FLAG-tagged proteins, expression remains under control of the gene's endogenous regulatory elements, preserving normal expression patterns and physiological relevance—unlike transgenic approaches that may lead to overexpression artifacts .

• Improved signal-to-noise ratio: Some FLAG antibodies demonstrate exceptional sensitivity with minimal background staining, particularly valuable when detecting proteins expressed at low levels .

What considerations are important when selecting between different FLAG antibody clones for specific applications?

Different FLAG antibody clones have distinct properties that affect their performance in various experimental contexts:

• Calcium dependency: The M1 antibody clone binds FLAG peptide only in the presence of bivalent metal cations (preferably Ca²⁺), which enables specific elution strategies using chelating agents .

• Detection sensitivity: Some clones, such as the 2H8 antibody, demonstrate significantly higher affinity, enabling detection of proteins expressed at low levels that might be missed by other FLAG antibodies. For example, 2H8 antibody has been shown to stain FLAG-tagged G-protein-coupled receptors (GPCRs) more strongly than commercially available antibodies in both flow cytometry and immunostaining experiments .

• Background signal: High-affinity clones like 2H8 can produce cleaner results with minimal non-specific background staining, which is particularly important for immunostaining applications in vitro and in vivo .

• Immunoprecipitation efficiency: Some high-sensitivity antibodies require substantially less material (as little as 10 ng) to effectively immunoprecipitate FLAG-tagged proteins from cell lysates .

• Host species compatibility: For mouse tissue immunohistochemistry, antibodies from goat (like anti-DDDDK polyclonal) can reduce background associated with using mouse primary antibodies on mouse tissues .

How can researchers optimize FLAG-tagged protein detection in immunohistochemistry of tissue samples?

For optimal immunohistochemical detection of FLAG-tagged proteins in tissue samples, researchers should consider the following validated methodology:

• Tissue preparation:

  • Properly fix tissues (e.g., with 10% neutral buffered formalin)

  • Process and embed in paraffin according to standard protocols

  • Section at appropriate thickness (typically 4-5 μm)

• Antigen retrieval technique:

  • Perform heat-induced antigen retrieval (e.g., with citrate buffer pH 6.0)

  • Optimize retrieval conditions for specific tissue types

• Blocking strategy:

  • Block endogenous peroxidase activity (e.g., with 3% hydrogen peroxide)

  • Use appropriate protein blocking (e.g., serum from the species of secondary antibody)

• Antibody selection and concentration:

  • For FLAG detection in mouse tissues, use goat anti-DDDDK polyclonal antibody (0.5 μg/ml has been validated)

  • Incubate for optimal time (60 minutes at room temperature has shown good results)

• Detection system:

  • Use biotinylated secondary antibody (e.g., donkey anti-goat for 30 minutes)

  • Apply avidin-biotin-HRP complex (e.g., Vector Elite ABC-HRP reagent)

  • Develop with appropriate substrate (e.g., metal-enhanced DAB for 5 minutes)

• Counterstaining and mounting:

  • Counterstain with Mayer's Hematoxylin

  • Dehydrate through alcohol series, clear in xylenes, and coverslip

• Controls:

  • Include wild-type (non-FLAG-tagged) tissues as negative controls

  • When available, compare with antibodies against the native protein on adjacent sections

What strategies exist for FLAG-tagged protein elution in affinity purification?

Two primary strategies have been established for eluting FLAG-tagged proteins while maintaining protein structure and function:

• Calcium-dependent elution:

  • Applicable when using the M1 antibody clone that binds FLAG in a calcium-dependent manner

  • Elution is accomplished using chelating agents (such as EDTA) that sequester calcium ions

  • Advantage: Gentle elution under non-denaturing conditions

  • Consideration: Requires calcium presence during binding phase

• Competitive elution:

  • Uses excess free FLAG peptide to compete for antibody binding sites

  • Tagged proteins are displaced from antibody without harsh conditions

  • Advantage: Versatile approach that works with most FLAG antibodies

  • Consideration: Requires synthesis or purchase of free FLAG peptide

  • Typical concentration: 100-200 μg/ml of FLAG peptide in elution buffer

Both strategies preserve protein integrity and function better than traditional harsh elution methods such as extreme pH or denaturing agents, making them particularly valuable for functional studies of purified proteins.

How does the sensitivity of FLAG detection compare between western blotting and immunohistochemistry?

The sensitivity of FLAG detection varies significantly between western blotting and immunohistochemistry, with important technical considerations for each:

Western blotting:

• Typically demonstrates higher sensitivity for detecting low abundance proteins

• FLAG-HRP conjugated antibodies can achieve detection at dilutions of 1:4000, allowing efficient protein detection with minimal antibody consumption

• In comparative studies, detection with anti-FLAG antibodies often provides superior sensitivity compared to antibodies against the native protein, as demonstrated in studies with RIPK3.3×FLAG

• Denatured proteins may expose the FLAG epitope more consistently

Immunohistochemistry:

• Sensitivity varies significantly based on tissue fixation, processing, and antigen retrieval

• Proteins expressed at very low levels may be undetectable by IHC but detectable by western blotting

• Spatial resolution allows detection in specific cell types that might be diluted in whole tissue lysates

• Studies with RIPK3.3×FLAG demonstrated that certain tissues (bone marrow) showed few positive cells by IHC but fell below detection limits in western blotting of whole tissue lysates

• Certain high-affinity antibodies like 2H8 have demonstrated sufficient sensitivity to detect FLAG-tagged GPCRs expressed in vivo in the small intestine of mice under control of tissue-specific promoters

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

When working with FLAG-tagged proteins, researchers commonly encounter the following challenges with corresponding solutions:

• High background staining:

  • Solution: Use higher dilutions of primary antibody, optimize blocking protocols, and consider using antibodies from species different from the tissue source (e.g., goat anti-DDDDK for mouse tissues)

  • Include appropriate negative controls (wild-type tissues) to confirm specificity

• Weak signal detection:

  • Solution: Consider high-affinity antibody clones like 2H8 that demonstrate superior sensitivity for detecting proteins expressed at low levels

  • Optimize antigen retrieval methods for immunohistochemistry applications

  • For western blotting, using FLAG-HRP conjugated antibodies can improve sensitivity with optimal dilutions (e.g., 1:4000)

• Inconsistent detection across tissue types:

  • Solution: Adjust tissue-specific fixation times and antigen retrieval methods

  • Consider protein expression levels in different tissues (e.g., RIPK3.3×FLAG was detectable in heart, lung, liver, thymus, and spleen but not bone marrow due to expression level differences)

• Difficulties in FLAG-tagged GPCR detection:

  • Solution: Consider specialized high-affinity antibodies like 2H8 that have demonstrated superior performance with these challenging membrane proteins

  • Optimize membrane protein extraction protocols for western blotting applications

How can researchers validate the specificity of FLAG antibody detection?

Validation of FLAG antibody specificity is critical for experimental rigor. Multiple complementary approaches can be employed:

• Parallel detection methods:

  • Compare FLAG-tag detection with direct protein detection using antibodies against the native protein on adjacent tissue sections

  • Both methods should yield similar staining patterns in tissues from knock-in animals expressing FLAG-tagged proteins

• Negative controls:

  • Assess wild-type (non-FLAG-tagged) tissues with the anti-FLAG antibody to confirm absence of non-specific staining

  • Include isotype control antibodies to identify potential background from non-specific antibody binding

• Comparative analysis:

  • When available, include tissues from gene knockout animals as negative controls

  • For example, RIPK3-deficient mice were used to validate anti-RIPK3 antibody specificity, which complemented FLAG-tag validation

• Molecular weight verification:

  • In western blotting, the FLAG-tagged protein should show the expected molecular weight shift compared to the untagged version

  • FLAG tag adds approximately 1 kDa to the protein molecular weight

What technical considerations are important when designing FLAG-tagged knock-in mice for protein expression studies?

When designing FLAG-tagged knock-in mice for protein expression studies, researchers should consider these critical factors:

• Tag insertion strategy:

  • C-terminal tagging (inserting FLAG sequence into the last coding exon ahead of the translation termination codon) preserves most protein functions while allowing detection

  • N-terminal tagging may interfere with signal peptides or cellular localization

  • Internal tagging requires careful domain analysis to avoid disrupting functional regions

• Tag configuration:

  • 3×FLAG tag provides enhanced detection sensitivity compared to single FLAG epitope

  • Multiple repeats must be designed with proper spacing to ensure epitope recognition

• Expression control:

  • Preserve endogenous regulatory elements to maintain physiologically relevant expression levels

  • This approach is distinct from transgenic expression of epitope-tagged cDNAs, which often results in protein overexpression and may not accurately reflect normal expression patterns

• Homozygosity considerations:

  • Generate homozygous knock-in animals (FLAG/FLAG) for enhanced detection sensitivity

  • Verify that homozygous tagging doesn't impact protein function or animal phenotype

• Detection strategy:

  • For immunohistochemistry in mouse tissues, goat anti-DDDDK antibodies reduce background compared to mouse-derived antibodies

  • Validate detection protocols with appropriate controls before extensive phenotypic analysis

How does the PAT3G6AT antibody compare to other FLAG antibodies in terms of performance characteristics?

The PAT3G6AT FLAG antibody demonstrates distinct performance characteristics compared to other commonly used FLAG antibodies:

This comparison demonstrates that researchers should select FLAG antibodies based on specific application requirements, with PAT3G6AT offering reliable performance for standard western blotting and immunoprecipitation applications.

What are the advantages and limitations of the FLAG tag system compared to other epitope tag systems?

The FLAG tag system offers distinct advantages and limitations compared to other common epitope tagging systems:

Advantages:

• Small size (8 amino acids): Minimally disrupts protein structure and function compared to larger tags

• Hydrophilic nature: Improves accessibility for antibody recognition and reduces interference with protein folding

• Flexible elution options: Enables both competitive elution with FLAG peptide and calcium-dependent elution strategies

• High sensitivity detection: Some FLAG antibodies (e.g., 2H8) demonstrate exceptional sensitivity for detecting proteins expressed at low levels

• Validated in multiple systems: Extensively characterized in cell culture, tissues, and in vivo models including knock-in mice

Limitations:

• Potential proteolytic cleavage: Contains an enterokinase-cleavage site that could lead to tag removal in certain contexts

• Multiple antibody clones: Different clones have varying properties, requiring careful selection for specific applications

• Calcium-dependency considerations: Some antibodies (M1) require calcium for binding, limiting their use in calcium-free buffers

• Background concerns in mouse tissues: Mouse-derived antibodies may produce higher background when used on mouse tissues, requiring alternative host antibodies

How might emerging technologies enhance the utility of FLAG-tagged proteins in research?

Several emerging technologies promise to expand the research applications of FLAG-tagged proteins:

• Super-resolution microscopy:

  • Enhanced spatial resolution will allow more precise localization of FLAG-tagged proteins within subcellular compartments

  • Multi-color imaging with orthogonal epitope tags can reveal protein complex formation at nanoscale resolution

• Proximity labeling approaches:

  • Combining FLAG tags with proximity labeling enzymes (BioID, APEX) could enable mapping of protein interaction networks in living cells

  • FLAG-based purification complements proximity labeling for proteomic analysis

• CRISPR-Cas9 genome editing:

  • Streamlined generation of FLAG knock-in models in diverse organisms beyond mice

  • Multiplex tagging of protein families to study related proteins simultaneously

• Single-cell proteomics:

  • FLAG-based detection could help quantify tagged proteins in individual cells to understand cellular heterogeneity

  • Combining with single-cell transcriptomics could reveal post-transcriptional regulation mechanisms

• Antibody engineering:

  • Development of even higher affinity FLAG antibodies for detection of extremely low abundance proteins

  • Novel recombinant antibody formats optimized for specific applications (nanobodies, single-chain antibodies)

These technological advances will likely expand the utility of the FLAG tag system beyond its current applications, particularly for in vivo studies and low-abundance protein detection.