IGSF3 Antibody, FITC conjugated

What is IGSF3 and what cellular functions does it have?

IGSF3 (Immunoglobulin Superfamily Member 3) is a membrane protein belonging to the immunoglobulin superfamily, located on chromosome 1p13.1. It contains a 3648-bp open reading frame encoding a protein with eight immunoglobulin domains. IGSF3 shows structural similarity to V7, a surface protein found on human leukocytes. Functionally, IGSF3 has been implicated in cancer progression, particularly hepatocellular carcinoma (HCC), where it activates the NF-κB signaling pathway to promote tumor growth, cell proliferation, migration, and invasion. The protein appears to significantly influence cell cycle progression by affecting levels of cyclins (A and E) and cyclin-dependent kinases (CDK1 and CDK2) .

What is the tissue distribution pattern of IGSF3?

IGSF3 demonstrates wide tissue distribution with particularly high expression in the placenta, kidney, and lungs. This expression pattern has been confirmed through multiple methodologies including immunohistochemistry, western blotting, and quantitative real-time PCR. In pathological contexts, IGSF3 shows significantly elevated expression in hepatocellular carcinoma tissues compared to adjacent non-tumor tissues, with expression levels positively correlating with tumor mass, TNM stage, and lymph node metastasis .

What are the validated applications for IGSF3 antibodies?

IGSF3 antibodies have been successfully utilized in multiple research applications. Based on available data, validated applications include:

• Western Blotting: Proven effective for detecting IGSF3 in human lung tissue and various cell lines under non-reducing conditions, typically revealing a band at approximately 200 kDa .

• Flow Cytometry: Successfully used to detect IGSF3 in cell lines such as A549 human lung carcinoma cells, making fluorophore-conjugated antibodies particularly valuable for this application .

• Immunohistochemistry: Used for both paraffin-embedded (IHC-P) and frozen (IHC-F) tissue sections to evaluate IGSF3 expression in normal and pathological tissue samples .

• Immunocytochemistry (ICC): Applied to detect IGSF3 in cultured cells, allowing for subcellular localization studies .

By drawing analogies with FITC-conjugated antibodies for other proteins, FITC-conjugated IGSF3 antibodies would be particularly valuable for flow cytometry and fluorescence microscopy applications .

What sample preparation methods optimize IGSF3 detection?

For optimal IGSF3 detection, preparation methods should be tailored to the specific application:

• Western Blotting: Protein extraction should be performed using RIPA buffer supplemented with 1% protease and 10% phosphatase inhibitors. Samples should be electrophoresed using 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Critically, IGSF3 detection is most successful under non-reducing conditions, suggesting that disulfide bonds are important for antibody recognition of the epitope .

• Flow Cytometry: Cells should be detached using non-enzymatic methods when possible to preserve surface epitopes. Standard fixation with 2-4% paraformaldehyde followed by permeabilization (if intracellular detection is required) is recommended .

• Immunohistochemistry: For formalin-fixed, paraffin-embedded tissues, antigen retrieval methods should be optimized, typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). For frozen sections, acetone fixation for 10 minutes at -20°C typically yields good results while preserving antigenic epitopes .

What are the recommended dilutions for different applications of IGSF3 antibodies?

While specific dilutions for IGSF3 antibodies must be optimized for each antibody formulation and application, the following ranges serve as general guidelines based on similar antibody applications:

• Western Blotting: 1 μg/mL has been successfully used for detecting IGSF3 in human lung tissue lysates. For other applications, dilutions ranging from 1:300-1:5000 may be appropriate, depending on the specific antibody and sample type .

• Immunohistochemistry/Immunofluorescence: Dilutions between 1:50-1:200 are typically appropriate for both paraffin-embedded and frozen sections .

• Flow Cytometry: Starting with a concentration similar to that used for immunohistochemistry (approximately 1:100) is recommended, with subsequent optimization based on signal-to-noise ratio .

Each laboratory should determine optimal dilutions empirically for their specific experimental conditions and antibody lots .

What are the advantages of using FITC-conjugated IGSF3 antibodies?

FITC-conjugated antibodies offer several advantages for IGSF3 detection:

• Direct Detection: FITC conjugation eliminates the need for secondary antibodies, reducing protocol complexity, time, and potential cross-reactivity issues.

• Multiplexing Capability: FITC (emission ~520 nm) can be combined with other fluorophores that have distinct spectral profiles for simultaneous detection of multiple targets in the same sample.

• Quantitative Analysis: Particularly valuable for flow cytometry applications, where fluorescence intensity correlates with antigen expression levels, allowing for quantitative assessment of IGSF3 expression.

• Reduced Background: Direct conjugation can reduce non-specific binding often associated with secondary detection systems .

What are optimal storage conditions for FITC-conjugated antibodies?

FITC-conjugated antibodies require specific storage conditions to maintain fluorophore activity:

• Temperature: Store at -20°C, protected from light. The inclusion of 50% glycerol in storage buffer prevents freeze-thaw damage.

• Aliquoting: Divide into multiple small aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade both antibody function and fluorophore activity.

• Buffer Composition: Optimal storage buffer typically contains 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300 (or similar preservative), and 50% Glycerol.

• Light Protection: Always store in amber tubes or wrapped in aluminum foil to protect the fluorophore from photobleaching .

How do I optimize signal-to-noise ratio when using FITC-conjugated IGSF3 antibodies?

To achieve optimal signal-to-noise ratios with FITC-conjugated IGSF3 antibodies:

• Titrate Antibody Concentration: Systematically test a range of antibody dilutions to determine the optimal concentration that maximizes specific signal while minimizing background.

• Include Proper Controls: Always include unstained controls, isotype controls (e.g., FITC-conjugated IgG matching the host species and isotype of the IGSF3 antibody), and known positive and negative controls.

• Optimize Fixation and Permeabilization: Excessive fixation can mask epitopes, while inadequate fixation can alter cellular morphology. Similarly, permeabilization conditions affect antibody access to intracellular epitopes.

• Use Blocking Solutions: Pre-incubation with appropriate blocking solutions (containing serum from the same species as the secondary antibody or BSA) reduces non-specific binding.

• Washing Stringency: Implement sufficient washing steps using appropriate buffers (PBS with 0.05-0.1% Tween-20) to remove unbound antibody .

How can I use IGSF3 antibodies to study its role in the NF-κB signaling pathway?

IGSF3 has been shown to promote hepatocellular carcinoma progression through activation of the NF-κB pathway. To investigate this relationship:

• Combinatorial Immunostaining: Use FITC-conjugated IGSF3 antibodies alongside antibodies against NF-κB pathway components (p65, phosphorylated IKBα) labeled with spectrally distinct fluorophores to visualize their spatial relationships in cells.

• Knockdown/Overexpression Studies: Manipulate IGSF3 expression using siRNA, shRNA, or overexpression plasmids, then assess NF-κB pathway activation through:

  • Western blot analysis of p65 nuclear translocation

  • Immunofluorescence localization of p65

  • Measurement of IKBα phosphorylation and degradation

  • NF-κB reporter assays

• Reversal Experiments: After IGSF3 knockdown, attempt to rescue the phenotype using TNF-α treatment, which activates the NF-κB pathway downstream of IGSF3 .

How does IGSF3 expression correlate with hepatocellular carcinoma progression?

IGSF3 expression shows significant clinical correlations with HCC progression:

What functional assays can demonstrate the biological effects of IGSF3?

To investigate IGSF3's functional roles, several validated assays have been employed:

• Proliferation Assays:

  • WST-1 assay shows reduced proliferation in IGSF3-knockdown cells

  • Colony formation assays demonstrate impaired foci formation capacity

  • PCNA expression analysis by western blot

• Cell Cycle Analysis:

  • Flow cytometry revealing G1 phase accumulation in IGSF3-knockdown cells

  • Western blot analysis of cyclins (A and E) and CDKs (1 and 2)

• Migration and Invasion Assays:

  • Transwell migration assays

  • Matrigel invasion assays

  • Wound-healing tests showing abrogated migration in IGSF3-silenced cells

• In Vivo Tumor Growth:

  • Mouse xenograft models showing reduced tumor growth with IGSF3 knockdown

Why might I see weak or no signal when using IGSF3 antibodies in western blotting?

Several factors can contribute to poor signal detection in western blotting:

• Reducing vs. Non-reducing Conditions: IGSF3 detection appears to work best under non-reducing conditions, suggesting that disulfide bonds are important for antibody epitope recognition. Always verify the recommended conditions for your specific antibody .

• Antibody Concentration: IGSF3 is detected optimally at approximately 1 μg/mL in western blotting of human lung tissue. Inadequate antibody concentration may result in weak signals .

• Buffer Compatibility: Use appropriate immunoblot buffer groups (e.g., Immunoblot Buffer Group 1 has been validated for IGSF3 detection) .

• Molecular Weight Verification: IGSF3 typically appears at approximately 200 kDa, significantly higher than its calculated molecular weight of 135.2 kDa, likely due to glycosylation and other post-translational modifications .

• Sample Preparation: Ensure complete protein extraction using RIPA buffer with appropriate protease and phosphatase inhibitors to prevent degradation during processing .

How can I improve IGSF3 detection in flow cytometry experiments?

To enhance IGSF3 detection in flow cytometry:

• Cell Preparation: Gentle cell dissociation methods help preserve surface epitopes. For adherent cells like A549, use non-enzymatic cell dissociation solutions or mild enzymatic methods followed by recovery periods.

• Antibody Selection: Use antibodies specifically validated for flow cytometry. For example, Sheep Anti-Human IGSF3 Antigen Affinity-purified Polyclonal Antibody has been successfully used to detect IGSF3 in A549 human lung carcinoma cells .

• Secondary Antibody Optimization: When using unconjugated primary antibodies, select appropriate fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody has been validated for IGSF3 detection) .

• Controls: Include appropriate isotype controls (e.g., control sheep IgG) to establish gating strategies and distinguish specific from non-specific binding .

What are the critical considerations for immunohistochemical detection of IGSF3?

For successful immunohistochemical detection of IGSF3:

• Tissue Preparation: Proper fixation and processing are critical; overfixation can mask epitopes. For IGSF3, both frozen and paraffin-embedded tissues can be used with appropriate preparation protocols.

• Antigen Retrieval: For FFPE tissues, heat-induced epitope retrieval methods improve accessibility of IGSF3 epitopes that may be masked during fixation and embedding processes.

• Dilution Optimization: Antibody concentrations between 1:50-1:200 are typically effective for immunohistochemical applications, but should be optimized for each specific antibody and tissue type .

• Counterstaining: Nuclear counterstains provide context for interpreting IGSF3 membrane localization. For fluorescence applications, DAPI is commonly used, while hematoxylin works well for chromogenic detection.

• Validation: Compare results with known expression patterns. IGSF3 shows particularly high expression in placenta, kidney, and lung tissues, which can serve as positive controls .