RASSF8 Antibody

What is RASSF8 and why is it significant in cancer research?

RASSF8 (Ras association domain family member 8) is an evolutionarily conserved member of the N-terminal RASSF family, which also includes RASSF7, PAMCI (RASSF9), and RASSF10. This protein contains an N-terminal RA (Ras association) domain and participates in the Ras signaling pathway . Its significance in cancer research stems from its tumor suppressor properties, particularly in:

• Melanoma: RASSF8 expression decreases with melanoma progression, with lower expression observed in metastatic melanoma compared to primary melanomas

• Lung cancer: Ectopic expression of RASSF8 inhibits anchorage-independent growth of lung cancer cells

• Cell regulation: RASSF8 inhibits cell growth, migration, and invasion while inducing apoptosis through the P53-P21 pathway

Research has demonstrated that RASSF8 exerts its tumor suppressive function by regulating P65 expression and its downstream target IL-6, thereby controlling tumor cell growth, migration, and invasion .

What are the typical detection methods and applications for RASSF8 antibodies?

RASSF8 antibodies have been validated for multiple research applications, including:

RASSF8 antibodies have been successfully used to demonstrate that RASSF8 expression is lower in stage IV melanomas compared to earlier stages, correlating with patient survival rates .

How can researchers verify the specificity of RASSF8 antibodies?

Verifying antibody specificity is critical for reliable results. For RASSF8 antibodies, consider these validation approaches:

• Positive and negative control samples:

  • Use cell lines with known RASSF8 expression (e.g., Wm266-4 RASSF8 as positive control)

  • Use RASSF8 knockdown samples (e.g., M24 RASSF8 shRNA) as negative controls

• Immunofluorescence validation:

  • Perform IF staining in known RASSF8-positive cells and RASSF8-knockdown cells

  • Compare staining patterns and intensity (as demonstrated in supplementary figures 3A and 3B in the literature)

• Western blot analysis:

  • Look for a single band at the expected molecular weight (~46 kDa)

  • Compare with expression patterns across different cell types (e.g., metastatic vs. primary melanoma lines)

• siRNA/shRNA knockdown validation:

  • Demonstrate reduction in signal following RASSF8 knockdown

  • This confirms that the antibody is detecting the intended target

What sample preparation protocols optimize RASSF8 detection in immunohistochemistry?

For optimal detection of RASSF8 in tissue samples:

• Fixation:

  • Standard formalin fixation and paraffin embedding (FFPE) is suitable

  • Avoid overfixation which may mask epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval is generally recommended

  • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions

• Blocking:

  • Use appropriate blocking solution to reduce background (e.g., 5% normal serum from the same species as the secondary antibody)

  • Include peroxidase blocking if using HRP-based detection systems

• Antibody concentration:

  • Titrate antibody to determine optimal dilution (typically 1:200-1:500)

  • Include isotype controls to assess non-specific binding

• Detection method:

  • For weak expression, consider polymer-based or tyramide signal amplification methods

  • DAB or other chromogens can be used for visualization

This approach has been successful in revealing RASSF8 expression patterns in melanoma tissue microarrays, demonstrating significantly lower RASSF8 expression in AJCC stage IV melanomas compared to earlier stages .

How can RASSF8 antibodies be employed to investigate its role in the P53-P21 signaling pathway?

RASSF8 induces apoptosis by activating the P53-P21 pathway. To investigate this relationship:

• Co-immunoprecipitation studies:

  • Use RASSF8 antibodies to pull down protein complexes

  • Perform western blot analysis to detect associated proteins (P53, P21)

  • This can reveal direct or indirect interactions between RASSF8 and pathway components

• Dual immunofluorescence staining:

  • Co-stain cells with RASSF8 and P53/P21 antibodies

  • Assess colocalization using confocal microscopy

  • Analyze changes in expression patterns following treatments

• Pathway analysis:

  • Use RASSF8 overexpression or knockdown models

  • Monitor changes in P53, P21, and Cyclin D1 expression by western blot

  • Research has shown that RASSF8 induces P53 and P21 expression while downregulating Cyclin D1

• Apoptosis assays:

  • Measure caspase activity in cells with varying RASSF8 expression levels

  • RASSF8 overexpression significantly increases caspase activity in Wm266-4 cells

• Human apoptosis array analysis:

  • Screen for changes in apoptosis-related proteins

  • Studies have shown RASSF8 overexpression induces P21 and TRAIL R2/DR5

What strategies can researchers employ to investigate RASSF8 methylation status in relation to protein expression?

RASSF8 expression is regulated by promoter methylation, particularly in melanoma progression. To investigate this relationship:

• Methylation-specific PCR (MS-PCR):

  • Design primers specific to methylated and unmethylated regions of the RASSF8 promoter

  • Use bisulfite-converted DNA from samples of different cancer stages

  • Higher methylation levels have been observed in stages III and IV melanoma compared to primary tumors (stages I/II)

• Correlation analysis:

  • Perform MS-PCR to determine methylation status

  • Use RASSF8 antibodies for IHC or western blot to assess protein expression

  • Analyze correlation between methylation and expression levels

• Demethylation studies:

  • Treat cell lines with 5-aza-2'-deoxycytidine (5-aza)

  • Use RASSF8 antibodies to assess protein re-expression by western blot

  • Treatment with 5-aza has been shown to induce RASSF8 expression

• TCGA data integration:

  • Analyze TCGA datasets for RASSF8 methylation in different cancer stages

  • Significantly higher hypermethylation of RASSF8 has been observed in metastatic melanoma tissues compared to earlier stage tumors

• Methylation-expression heatmaps:

  • Create visual representations of the inverse relationship between methylation and expression

  • Use this to demonstrate the epigenetic regulation of RASSF8

What are common challenges in detecting RASSF8 protein and how can they be addressed?

Researchers may encounter several challenges when working with RASSF8 antibodies:

• Low endogenous expression:

  • Problem: RASSF8 expression is often low, particularly in metastatic cells

  • Solution: Use signal amplification methods (e.g., TSA), increase antibody concentration, or extend incubation times

  • Consider using cell lines with known high RASSF8 expression as positive controls

• Non-specific binding:

  • Problem: Multiple bands or diffuse staining

  • Solution: Optimize blocking conditions, increase washing stringency, and titrate antibody concentration

  • Use RASSF8 knockdown controls to confirm specificity

• Subcellular localization variability:

  • Problem: RASSF8 localizes to both nucleus and cytoplasm , making interpretation challenging

  • Solution: Use confocal microscopy for precise localization and co-stain with nuclear and cytoplasmic markers

• Cross-reactivity with other RASSF family members:

  • Problem: Potential cross-reactivity with related proteins

  • Solution: Validate with specific controls (overexpression/knockdown) and consider epitope mapping

  • Use antibodies raised against unique regions of RASSF8

• Inconsistent fixation effects:

  • Problem: Variable results between frozen and FFPE samples

  • Solution: Standardize fixation protocols and optimize antigen retrieval for FFPE samples

  • Test multiple antibody clones if available

How can RASSF8 antibodies be optimized for dual-staining experiments with P65 or P53?

For successful dual immunofluorescence studies investigating RASSF8 interactions:

• Antibody compatibility:

  • Select primary antibodies from different host species (e.g., rabbit anti-RASSF8 and mouse anti-P65)

  • If using same-species antibodies, consider sequential staining with direct-labeled antibodies

• Signal separation:

  • Choose fluorophores with minimal spectral overlap

  • Include proper compensation controls if using flow cytometry

  • Use spectral unmixing for confocal microscopy if needed

• Optimization protocol:

  • Test antibodies individually before combining

  • Optimize concentration of each antibody separately

  • Determine optimal blocking to minimize background

  • Consider tyramide signal amplification for weak signals

• Controls for co-localization studies:

  • Include single-stained controls

  • Use cells with RASSF8 knockdown as negative controls

  • Include positive controls where co-localization is expected

• Quantitative analysis:

  • Use appropriate software (ImageJ, CellProfiler) for co-localization analysis

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Analyze multiple fields and cells for statistical significance

This approach can help investigate the relationship between RASSF8 and P65, as research has shown RASSF8 regulates P65 expression and its downstream target IL-6 .

How can RASSF8 antibodies contribute to understanding its role in in vivo tumor models?

RASSF8 antibodies can be valuable tools for in vivo tumor model studies:

• Xenograft tumor analysis:

  • Use IHC with RASSF8 antibodies to analyze protein expression in tumors

  • Compare control tumors with those from RASSF8-modulated cells

  • Studies have shown RASSF8 knockdown enhances growth of xenograft tumors in nude mice

• Tumor growth monitoring:

  • Track RASSF8 expression changes during tumor progression

  • Correlate expression with tumor volume and weight

  • Analyze relationship with metastatic potential

• Pharmacological intervention studies:

  • Monitor RASSF8 expression following drug treatments

  • Use RASSF8 antibodies to assess treatment effects on signaling pathways

  • Correlate with changes in P65 and P53-P21 pathway components

• Patient-derived xenograft (PDX) models:

  • Characterize RASSF8 expression in PDX models

  • Compare expression patterns between original patient samples and PDX tumors

  • Assess stability of expression over multiple passages

• Metastasis studies:

  • Use RASSF8 antibodies to compare primary and metastatic lesions

  • Analyze relationship between RASSF8 expression and metastatic potential

  • Investigate correlations with patient survival (patients with high RASSF8 expression in stage IV melanoma show higher survival rates)

What approaches can be used to investigate RASSF8 interactions with other proteins in the Ras signaling pathway?

To study RASSF8's role in the Ras signaling network:

• Co-immunoprecipitation:

  • Use RASSF8 antibodies to pull down protein complexes

  • Identify binding partners through mass spectrometry or western blot

  • Validate interactions with reciprocal co-IP

• Proximity ligation assay (PLA):

  • Visualize protein-protein interactions in situ

  • Combine RASSF8 antibody with antibodies against potential binding partners

  • Quantify interaction signals at subcellular resolution

• FRET/BRET studies:

  • Generate fusion proteins (RASSF8-donor and partner-acceptor)

  • Measure energy transfer as indicator of protein proximity

  • Analyze dynamics of interactions in live cells

• Immunofluorescence co-localization:

  • Use dual staining with RASSF8 and Ras pathway component antibodies

  • Analyze co-localization using confocal microscopy

  • Quantify changes in response to pathway activation/inhibition

• Protein interaction domain mapping:

  • Generate RASSF8 deletion mutants lacking specific domains

  • Use antibodies that recognize remaining epitopes

  • Determine which domains are required for specific interactions

These approaches can help elucidate how RASSF8, through its N-terminal RA domain, participates in the Ras signaling pathway and exerts its tumor suppressor functions .