FUT13 Antibody

What is FUT13 and how does it relate to POFUT2?

FUT13 is a reported synonym of the POFUT2 gene, which encodes protein O-fucosyltransferase 2. This protein plays a significant role in the regulation of gene expression among other biological functions. The human version of FUT13/POFUT2 has a canonical amino acid length of 429 residues and a protein mass of 50 kilodaltons, with three distinct isoforms identified to date. It's important to note that in research literature and antibody catalogs, FUT13 and POFUT2 are often used interchangeably, with additional synonyms including C21orf80, fc46a11, wu:fc46a11, zgc:194822, and 2310011G23Rik .

What is the cellular localization of FUT13/POFUT2?

FUT13/POFUT2 is predominantly localized in the endoplasmic reticulum (ER) and Golgi apparatus within cells. This localization is consistent with its function as a glycosyltransferase involved in post-translational modifications of proteins. When designing immunofluorescence or subcellular fractionation experiments, researchers should expect to detect this protein primarily in these organelles, which may require specific cell permeabilization protocols to access these intracellular compartments .

How do FUT13 antibodies differ from other fucosyltransferase antibodies like FUT3?

While both target fucosyltransferase family proteins, FUT13/POFUT2 antibodies specifically recognize protein O-fucosyltransferase 2, whereas FUT3 antibodies target fucosyltransferase 3, which catalyzes the addition of fucose to precursor polysaccharides in the final step of Lewis antigen biosynthesis. FUT3 is involved in creating fucosylated glycosphingolipids that function in embryogenesis, tissue differentiation, tumor metastasis, inflammation, and bacterial adhesion. The distinct functional and structural differences between these proteins necessitate careful selection of antibodies with validated specificity to avoid cross-reactivity in experimental systems .

What criteria should researchers use when selecting a FUT13 antibody for their experiments?

When selecting a FUT13/POFUT2 antibody, researchers should consider:

• Specificity: Verify the antibody recognizes the specific epitope region of FUT13/POFUT2 (e.g., antibodies targeting AA 125-271 as mentioned in search results).

• Validated applications: Ensure the antibody is validated for your intended application (common applications include Western Blot, ELISA, and Immunohistochemistry).

• Species reactivity: Confirm the antibody reacts with your study species (human, mouse, rat, or Arabidopsis variants are available).

• Clonality: Decide between monoclonal (higher specificity) or polyclonal (potentially higher sensitivity) based on your experimental needs.

• Conjugation status: Determine if you need an unconjugated primary antibody or one conjugated to a detection molecule.

For critical research, evaluate literature citations and validation data provided by manufacturers before selection .

How can researchers validate the specificity of a FUT13 antibody?

To validate FUT13 antibody specificity, implement these methodological approaches:

• Positive and negative controls: Use tissue/cells known to express high levels of FUT13/POFUT2 as positive controls and those lacking expression as negative controls.

• Knockdown/knockout validation: Test the antibody on samples where FUT13/POFUT2 has been silenced via siRNA or CRISPR to confirm signal reduction.

• Preabsorption test: Preincubate the antibody with purified FUT13/POFUT2 protein before immunostaining to verify signal reduction.

• Multiple antibody comparison: Use different antibodies targeting distinct epitopes of FUT13/POFUT2 to confirm consistent detection patterns.

• Mass spectrometry validation: Confirm protein identity in immunoprecipitated samples using mass spectrometry.

Thorough validation is essential, particularly for lesser-studied proteins like FUT13, to ensure experimental observations are attributable to the target protein .

What are the optimal conditions for using FUT13 antibodies in Western blot applications?

For optimal Western blot detection of FUT13/POFUT2:

• Sample preparation: Extract proteins using buffers containing appropriate detergents (RIPA or NP-40) that can solubilize ER/Golgi membrane proteins.

• Protein loading: Load 20-50μg of total protein per lane (may require optimization).

• Gel percentage: Use 10% SDS-PAGE gels to properly resolve the ~50kDa FUT13/POFUT2 protein.

• Transfer conditions: Transfer to PVDF membranes at 100V for 1 hour or 30V overnight in 20% methanol transfer buffer.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute antibody 1:500-1:2000 (optimize based on specific antibody) and incubate overnight at 4°C.

• Secondary antibody incubation: Use appropriate HRP-conjugated secondary antibody at 1:5000-1:10000 for 1 hour at room temperature.

• Expected molecular weight: Look for primary band at approximately 50kDa, with possible additional bands for isoforms.

These conditions should be optimized for each specific antibody and sample type .

How should researchers design immunohistochemistry experiments using FUT13 antibodies?

For successful immunohistochemistry with FUT13/POFUT2 antibodies:

• Fixation: Use 4% paraformaldehyde or 10% neutral buffered formalin; avoid over-fixation which can mask epitopes.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to expose ER/Golgi-localized epitopes.

• Permeabilization: Include a membrane permeabilization step (0.1-0.5% Triton X-100) to access intracellular compartments.

• Blocking: Block with 5-10% normal serum from the species of secondary antibody for 1 hour.

• Primary antibody incubation: Dilute FUT13 antibody 1:100-1:500 and incubate overnight at 4°C.

• Controls: Include technical controls (no primary antibody) and biological controls (tissues known to express/not express FUT13).

• Signal detection: Use appropriate detection systems (HRP/DAB or fluorescent) depending on desired sensitivity and multiplexing needs.

• Expected staining pattern: Look for perinuclear staining consistent with ER/Golgi localization.

Optimize these parameters based on tissue type and specific antibody characteristics .

How can FUT13 antibodies be utilized to investigate O-fucosylation in protein regulation?

To investigate O-fucosylation using FUT13/POFUT2 antibodies:

• Co-immunoprecipitation studies: Use FUT13 antibodies to pull down protein complexes, then analyze interacting partners to identify substrate proteins.

• Proximity ligation assays: Combine FUT13 antibodies with antibodies against putative substrate proteins to visualize and quantify protein interactions in situ.

• Mass spectrometry workflow:

  • Immunoprecipitate FUT13/POFUT2 under native conditions to maintain enzyme-substrate interactions

  • Perform LC-MS/MS analysis to identify fucosylated peptides

  • Use collision-induced dissociation techniques to map specific O-fucosylation sites

• Comparative analysis: Compare fucosylation patterns in wild-type versus FUT13/POFUT2 knockdown/knockout models to identify specific substrates.

• Functional assays: Correlate changes in protein function with O-fucosylation status using activity assays before and after manipulation of FUT13/POFUT2 levels.

This multilayered approach can provide insights into how FUT13-mediated O-fucosylation regulates target protein function and cellular processes .

What approaches can be used to develop novel FUT13 antibodies with improved specificity and sensitivity?

Advanced approaches for developing improved FUT13/POFUT2 antibodies include:

• Rational epitope selection:

  • Target unique regions of FUT13/POFUT2 with low homology to other fucosyltransferases

  • Focus on accessible regions based on protein structure prediction

  • Consider functional domains that may have structural stability

• Alternative production methods:

  • Utilize yeast display systems with fluorescent-labeled target proteins for antibody screening without animal immunization

  • Employ phage display libraries to select high-affinity antibody fragments

  • Consider synthetic antibody libraries with designed complementarity determining regions

• Validation strategies:

  • Implement CRISPR-edited cell lines expressing tagged FUT13/POFUT2 for validation

  • Use multiple orthogonal techniques (WB, IP-MS, IF) to confirm specificity

  • Perform cross-reactivity testing against related fucosyltransferases

• Format optimization:

  • Develop recombinant antibody fragments (Fab, scFv) for improved tissue penetration

  • Create bispecific antibodies targeting FUT13 and its substrates simultaneously

These strategies can overcome limitations of traditional antibody production methods while improving specificity for challenging targets like FUT13 .

What are common challenges when using FUT13 antibodies and how can they be addressed?

Systematic troubleshooting using this framework can identify specific issues in FUT13 antibody applications and guide targeted solutions .

How can researchers design competition assays to verify FUT13 antibody specificity?

To implement rigorous competition assays for FUT13/POFUT2 antibody validation:

• Peptide competition:

  • Pre-incubate the FUT13 antibody with varying concentrations (1-100μg/ml) of the immunizing peptide

  • Process paired samples (with/without peptide competition) using identical protocols

  • A specific antibody will show dose-dependent signal reduction with the competing peptide

• Recombinant protein competition:

  • Express and purify full-length or fragment recombinant FUT13/POFUT2

  • Pre-incubate antibody with increasing molar ratios of recombinant protein

  • Compare signal intensity across competition conditions

• Quantitative analysis:

  • Plot signal intensity versus competitor concentration

  • Calculate IC50 values for antibody-epitope interaction

  • Compare competition profiles between target peptide and unrelated control peptides

• Cross-competition with multiple antibodies:

  • Test competition between different FUT13 antibodies targeting distinct epitopes

  • Non-overlapping epitope antibodies should not compete, while overlapping epitope antibodies will

These approaches provide robust verification of antibody specificity while generating valuable binding kinetics data .

What methodological considerations are important when generating new monoclonal antibodies against FUT13?

When generating new monoclonal antibodies against FUT13/POFUT2, researchers should consider:

• Antigen design strategies:

  • Use purified recombinant protein or synthetic peptides from unique regions

  • Consider protein fragments (AA 125-271) that have shown success in previous antibodies

  • Ensure proper protein folding if conformational epitopes are desired

• Immunization protocol optimization:

  • Select appropriate mouse strain (BALB/c is most common for hybridoma generation)

  • Follow a structured immunization schedule:

    • Primary immunization with Complete Freund's Adjuvant (CFA)

    • Boosters at days 14 and 28 with Incomplete Freund's Adjuvant (IFA)

    • Titer testing at day 36

    • Final intravenous boost at day 56 without adjuvant

    • Spleen harvest and fusion at day 59

• Antibody screening considerations:

  • Design screening assays that match intended applications

  • Include counter-screening against related fucosyltransferases

  • Verify both sensitivity and specificity early in selection

• Production alternatives:

  • Consider in vitro production methods before ascites production

  • Explore yeast display systems as animal-free alternatives

  • Optimize culture conditions for hybridoma stability and yield

Following these methodological considerations increases the likelihood of generating high-quality, specific FUT13 antibodies suitable for research applications .