FUT5 Antibody

What is FUT5 and why is it important in research?

FUT5 (Fucosyltransferase 5) is an alpha-1,3-fucosyltransferase enzyme involved in the synthesis and glycosylation of various proteins. Research has shown that FUT5 plays critical roles in multiple biological processes, including cancer progression. Specifically, FUT5 has been found to be upregulated in intrahepatic cholangiocarcinoma (ICC) and promotes ICC development through glycosylation of several proteins including versican, β3 integrin, and cystatin 7 . Understanding FUT5's functions provides insights into disease mechanisms and potential therapeutic targets.

What types of FUT5 antibodies are available for research?

Researchers have access to both monoclonal and polyclonal FUT5 antibodies from various providers:

These antibodies have been validated through multiple assays to ensure reliability for research applications .

How should I select the appropriate FUT5 antibody for my research?

When selecting a FUT5 antibody, consider:

• Target epitope - Different antibodies target different amino acid regions of the FUT5 protein. For example, some target AA 95-164 while others target AA 281-374 . Choose based on your region of interest.

• Application compatibility - Verify the antibody has been validated for your intended application (WB, IHC, IF, ELISA, etc.).

• Host species - Consider potential cross-reactivity with your experimental system.

• Validation status - Prefer antibodies with enhanced validation through orthogonal or independent antibody methods .

• Clonality - Monoclonal antibodies offer greater specificity and reproducibility, while polyclonal antibodies may provide stronger signals and recognize multiple epitopes .

What are the optimal protocols for using FUT5 antibodies in immunohistochemistry?

For optimal immunohistochemistry results with FUT5 antibodies:

• Sample preparation: Use paraffin-embedded tissues that are properly deparaffinized, rehydrated, and rinsed with distilled water.

• Antigen retrieval: Prepare human FUT5 antigen according to standard protocols.

• Primary antibody incubation: Incubate slides overnight with anti-FUT5 polyclonal antibody at a 1:200 dilution at 4°C.

• Secondary antibody and staining: Incubate with appropriate secondary antibodies and stain with diaminobenzidine.

• Evaluation: Assess staining intensity by randomly selecting 5 non-overlapping fields at 40× magnification for each slide and quantify by calculating the percentage of FUT5-positive cells .

This protocol has been successfully employed to demonstrate FUT5 upregulation in ICC tissues compared to adjacent non-tumor tissues .

How can I optimize Western blotting protocols for FUT5 detection?

For optimal Western blotting results with FUT5 antibodies:

• Protein extraction: Use standard lysis buffers containing protease inhibitors to extract total protein from cells or tissues.

• Sample preparation: Load 20-30 μg of protein per lane.

• Antibody dilution: Use anti-FUT5 antibody at a 1:1000 dilution for best results.

• Controls: Include appropriate positive controls (tissues known to express FUT5) and loading controls (GAPDH at 1:2000 dilution).

• Incubation time: Overnight incubation with primary antibody at 4°C typically yields optimal results.

• Detection: Use appropriate HRP-conjugated secondary antibodies followed by ECL detection .

This approach has successfully been used to validate FUT5 knockdown and overexpression in functional studies of ICC cells .

What controls should I include when using FUT5 antibodies?

When working with FUT5 antibodies, include the following controls:

• Positive controls: Cell lines with verified FUT5 expression (e.g., HuCCT1 cells have been shown to express higher levels of FUT5 compared to HCCC-9810 and RBE cells) .

• Negative controls: Either tissues/cells known not to express FUT5 or samples treated with negative control shRNA.

• Isotype controls: For immunostaining, include appropriate isotype-matched control antibodies.

• Loading controls: For Western blotting, use antibodies against housekeeping proteins like GAPDH (1:2000) or vinculin (1:1000) .

• Knockdown validation: When possible, include samples with confirmed FUT5 knockdown using validated shRNA sequences .

How can I study the role of FUT5 in cancer progression?

To investigate FUT5's role in cancer progression, consider these methodological approaches:

• Expression analysis: Compare FUT5 expression in tumor versus normal tissues using immunohistochemistry and qPCR. In ICC studies, FUT5 was significantly upregulated in tumor tissues .

• Functional studies through genetic manipulation:

  • Overexpression: Clone FUT5 coding sequence into appropriate vectors (e.g., CV146) and establish stable cell lines using lentiviral transduction followed by puromycin selection .

  • Knockdown: Design FUT5-specific shRNAs (validated sequences available) and generate stable cell lines .

• Proliferation assays: Employ CCK8 assays (1000 cells/well, measured daily for 4 days) to assess the impact of FUT5 on cell proliferation .

• Migration assays: Use Transwell migration assays to evaluate FUT5's effect on cell migratory capacity .

• Colony formation assays: Seed 1000 cells/well in 6-well plates and culture for 10 days to assess long-term proliferative potential .

• Mechanistic studies: Investigate downstream targets and pathways affected by FUT5 using proteomics approaches .

These approaches have revealed that FUT5 enhances proliferation and migration in ICC cells, providing valuable insights into its role in cancer progression .

What methodologies can I use to study FUT5-mediated protein glycosylation?

To investigate FUT5-mediated protein glycosylation:

• Identify target proteins: Research has shown that FUT5 is essential for the glycosylation of several proteins, including versican, β3 integrin, and cystatin 7 .

• Protein interaction studies: Use co-immunoprecipitation with FUT5 antibodies to identify proteins that interact with FUT5.

• Glycoprotein analysis:

  • Western blotting using antibodies against specific glycoproteins (e.g., ITGB3 at 1:3000 dilution) .

  • Lectin binding assays to detect specific glycan structures.

• Functional consequences: Assess the impact of altered glycosylation on protein function, cellular localization, and signaling pathways.

• Comparative analysis: Compare glycosylation patterns between FUT5-overexpressing and FUT5-knockdown cells to identify FUT5-specific modifications.

This integrated approach can help determine how FUT5-mediated glycosylation influences protein function and cellular phenotypes in normal and disease states .

How can I address discrepancies in FUT5 expression data between different detection methods?

When facing discrepancies in FUT5 expression data:

• Antibody validation assessment:

  • Verify antibody specificity through orthogonal validation and independent antibody validation .

  • Check if the antibodies target different epitopes (e.g., AA 95-164 vs. AA 281-374) .

  • Confirm antibody validation scores from reliable sources (Enhanced, Supported, Approved, or Uncertain) .

• Methodological considerations:

  • mRNA vs. protein expression: Assess if discrepancies exist between transcriptional and translational levels.

  • Sensitivity differences: Different techniques have varying detection thresholds.

  • Sample preparation variations: Fixation and processing can affect epitope accessibility.

• Biological considerations:

  • Tissue heterogeneity: FUT5 expression may vary within tissues, as observed in ICC samples showing both cellular and extracellular matrix localization .

  • Subcellular localization: Consider if different methods detect FUT5 in different cellular compartments.

• Reconciliation strategies:

  • Use multiple antibodies targeting different epitopes

  • Employ complementary techniques (IHC, WB, qPCR)

  • Include appropriate controls to validate each method

What are common challenges when working with FUT5 antibodies and how can they be addressed?

Researchers frequently encounter these challenges when working with FUT5 antibodies:

• Background signal:

  • Optimize antibody dilution (start with 1:200 for IHC, 1:1000 for WB)

  • Increase washing steps and duration

  • Use appropriate blocking agents (5% BSA or milk)

  • Consider using monoclonal antibodies for higher specificity

• Inconsistent results:

  • Store antibodies according to manufacturer recommendations (e.g., 50% glycerol, -20°C)

  • Aliquot antibodies to avoid repeated freeze-thaw cycles

  • Standardize sample preparation and experimental conditions

  • Use validated positive controls like HuCCT1 cells

• Lack of signal:

  • Verify target expression in your sample (FUT5 expression varies across tissues and cell lines)

  • Optimize antigen retrieval methods for IHC

  • Consider more sensitive detection systems

• Cross-reactivity:

  • Check for sequence homology with other fucosyltransferases

  • Validate specificity through knockdown experiments using validated shRNA sequences

What factors should be considered when validating novel findings about FUT5 function?

When validating novel findings about FUT5 function:

What are the latest research directions involving FUT5 antibodies in cancer research?

Recent research with FUT5 antibodies is advancing our understanding of cancer biology in several ways:

These research directions highlight FUT5's potential as both a biomarker and therapeutic target in cancer research .

How can FUT5 antibodies be utilized to advance glycobiology research?

FUT5 antibodies offer valuable tools for advancing glycobiology research through:

These applications can significantly advance our understanding of glycobiology and its implications in health and disease.