SEMA4F Antibody, FITC conjugated

What is SEMA4F and what biological roles does it play?

SEMA4F is a transmembrane protein belonging to the class 4 semaphorin family. It plays significant regulatory functions in cancer biology, particularly showing prognostic value in gastric cancer as evidenced by recent studies . SEMA4F has a calculated molecular weight of approximately 84 kDa, though it is typically observed at 83 kDa in experimental conditions . The protein is encoded by the SEMA4F gene (GeneID: 10505) and is referenced by UniProt accession O95754 . Like other semaphorins, SEMA4F likely participates in cell migration, tissue organization, and potentially immune system regulation, though its specific functions are still being elucidated through ongoing research.

What applications are suitable for FITC-conjugated SEMA4F antibodies?

FITC-conjugated SEMA4F antibodies are particularly valuable for applications requiring fluorescent detection, with flow cytometry being the primary application. Based on validation data, these antibodies can be effectively used in:

• Flow cytometry analysis of cells expressing SEMA4F (such as Daudi cells)

• Immunofluorescence microscopy of cultured cells and tissue sections

• Fluorescent immunohistochemistry

• Multiplex immunostaining when combined with other fluorophore-conjugated antibodies

Unconjugated versions of the antibody have been validated for ELISA, western blotting, and standard immunohistochemistry, indicating the broader utility of SEMA4F antibodies across multiple experimental platforms .

What are the optimal sample types and preparation methods for SEMA4F detection?

SEMA4F antibodies have been validated across multiple sample types:

• Cell lines: Successful detection has been documented in U2OS cells for immunocytochemistry and Daudi cells for flow cytometry

• Tissue samples: SEMA4F antibodies have been effectively used for IHC analysis in:

  • Human colon adenocarcinoma

  • Human larynx squamous cell carcinoma

  • Human liver cancer

  • Human lung adenocarcinoma

For optimal results, tissue samples typically require heat-mediated antigen retrieval in EDTA buffer (pH 8.0), while cell preparations for flow cytometry benefit from fixation with 4% paraformaldehyde and blocking with 10% normal goat serum .

What controls should be included when working with FITC-conjugated SEMA4F antibodies?

When designing experiments using FITC-conjugated SEMA4F antibodies, the following controls are essential:

• Isotype control: Use rabbit IgG (matching the host species of the SEMA4F antibody) at the same concentration as the primary antibody (typically 1 μg per 1×10^6 cells for flow cytometry)

• Unlabeled control: Sample without incubation with primary antibody and secondary antibody to establish baseline autofluorescence

• Positive control: Samples known to express SEMA4F (e.g., Daudi cells have been validated)

• Negative control: Cell lines or tissues known not to express SEMA4F

• Secondary antibody-only control: For protocols using indirect detection methods

These controls help distinguish specific signals from background and non-specific binding, enabling accurate interpretation of results.

What are the recommended dilutions and concentrations for SEMA4F antibodies in different applications?

Based on validated protocols, the following dilutions and concentrations are recommended:

Optimal dilutions should be determined by the researcher through titration experiments for each specific application and sample type .

How should I optimize staining protocols for FITC-conjugated SEMA4F antibodies in flow cytometry?

For optimal flow cytometry results with FITC-conjugated SEMA4F antibodies:

• Cell preparation: Fix cells with 4% paraformaldehyde and block with 10% normal goat serum to reduce non-specific binding

• Antibody concentration: Start with 1 μg antibody per 1×10^6 cells

• Incubation conditions: 30 minutes at 20°C has been validated for Daudi cells

• Controls: Include isotype control (rabbit IgG) and unstained samples

• Compensation: If performing multicolor flow cytometry, proper compensation is essential to account for spectral overlap between FITC and other fluorophores

• Gating strategy: Begin with forward/side scatter gating to identify intact cells, followed by singlet gating before analyzing FITC signal

For direct conjugated antibodies, titration experiments to determine optimal concentration are recommended, as excessive antibody can increase background fluorescence.

How can I quantify SEMA4F expression in tumor tissues and correlate it with clinical outcomes?

To quantify SEMA4F expression in tumor tissues and correlate with clinical outcomes:

What could cause discrepancies between calculated and observed molecular weight of SEMA4F?

The calculated molecular weight of SEMA4F is 84 kDa, while the observed molecular weight is typically 83 kDa . Several factors could explain this discrepancy:

• Post-translational modifications:

  • Proteolytic processing removing small peptide fragments

  • Differential glycosylation patterns

  • Phosphorylation state differences

• Experimental variables:

  • Gel composition and running conditions affecting protein migration

  • Buffer systems and reducing conditions

  • Sample preparation methods (heating, reducing agents)

• Protein conformation:

  • More compact protein folding than predicted

  • Presence of intramolecular disulfide bonds affecting migration

These differences are common in protein research and typically do not indicate antibody specificity problems as long as they are consistent across experiments and validation studies.

How can SEMA4F antibodies contribute to cancer biomarker research?

SEMA4F antibodies offer significant potential for cancer biomarker research:

The ability to detect SEMA4F in various cancer tissues including colon adenocarcinoma, larynx squamous cell carcinoma, liver cancer, and lung adenocarcinoma suggests broad applicability across multiple cancer types .

What are the considerations for using SEMA4F antibodies in multiplex immunofluorescence?

When incorporating FITC-conjugated SEMA4F antibodies in multiplex immunofluorescence:

• Spectral characteristics:

  • FITC excites at ~495 nm and emits at ~519 nm (green fluorescence)

  • Plan panel design to avoid spectral overlap with other fluorophores

  • Consider pairing with fluorophores in distinctly different channels (e.g., Cy3, Cy5, APC)

• Antibody compatibility:

  • Ensure primary antibodies are from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential staining with proper blocking

  • Test antibodies individually before combining in multiplex panels

• Staining protocol optimization:

  • Adjust fixation and antigen retrieval methods to accommodate all targets

  • Titrate each antibody in the multiplex panel individually

  • Consider tyramide signal amplification for low-abundance targets

• Analysis considerations:

  • Use proper controls for spectral unmixing

  • Develop consistent thresholds for positivity

  • Consider colocalization analysis for protein interaction studies

Multiplex approaches can reveal important spatial relationships between SEMA4F and other proteins within the tumor microenvironment.

What could cause weak or absent staining with FITC-conjugated SEMA4F antibodies and how can these issues be resolved?

Several factors can contribute to weak or absent SEMA4F staining:

• Antibody-related issues:

  • Degradation due to improper storage (store at -20°C, avoid repeated freeze/thaw cycles)

  • Photobleaching of FITC fluorophore (minimize exposure to light)

  • Insufficient antibody concentration (optimize through titration)

• Sample preparation problems:

  • Inadequate fixation preserving antigen structure

  • Ineffective antigen retrieval (use EDTA buffer pH 8.0 as validated)

  • Overfixation masking epitopes

  • Sample degradation during storage

• Protocol optimization:

  • Insufficient blocking leading to high background (use 10% goat serum)

  • Inadequate incubation time (consider overnight at 4°C for primary antibody)

  • Suboptimal buffer composition

  • Inappropriate secondary antibody selection

Resolution strategies include:

• Titrating antibody concentration to determine optimal working dilution

• Testing alternative antigen retrieval methods

• Using signal amplification systems for low-abundance targets

• Confirming target expression through orthogonal methods (e.g., RT-qPCR)

• Verifying antibody functionality with positive control samples known to express SEMA4F

How can I properly store and handle FITC-conjugated antibodies to maintain activity?

To maintain optimal activity of FITC-conjugated SEMA4F antibodies:

• Storage conditions:

  • Store at -20°C in the dark

  • Prepare small aliquots to avoid repeated freeze/thaw cycles

  • Follow manufacturer's recommendations for buffer composition (typically PBS with 50% glycerol and 0.02% sodium azide)

• Handling practices:

  • Minimize exposure to light during all steps (FITC is photosensitive)

  • Keep on ice when in use

  • Return to -20°C promptly after use

  • Avoid prolonged incubation at room temperature

• Working solution preparation:

  • Dilute antibodies immediately before use

  • Use high-quality, filtered buffers

  • Consider adding protein stabilizers (BSA, gelatin) to working solutions

  • Check for microbial contamination

Properly stored and handled FITC-conjugated antibodies typically maintain activity for at least 6-12 months, though specific shelf-life should be confirmed with the manufacturer.