RASSF9 Antibody

What is the optimal application range for RASSF9 antibodies in immunohistochemistry?

RASSF9 antibodies typically perform best in immunohistochemistry at dilutions ranging from 1:50 to 1:200 . For optimal results, researchers should:

• Perform titration experiments to determine the ideal antibody concentration for specific tissue types

• Use positive control tissues with known RASSF9 expression (epidermis shows high expression levels)

• Implement antigen retrieval methods (heat-induced epitope retrieval at pH 9.0 is generally effective)

• Include negative controls (omitting primary antibody, isotype controls)

In skin tissue analysis, RASSF9-specific signals are detectable throughout the entire epidermis, with particularly strong expression in the suprabasal layer, as demonstrated by double immunofluorescence staining with K1 .

How can researchers validate the specificity of RASSF9 antibodies?

Antibody specificity validation should include multiple complementary approaches:

• Western blot analysis with recombinant RASSF9 protein (GST-tagged RASSF9 fusion proteins can serve as positive controls)

• Comparison between wild-type and RASSF9-knockout tissues (immunofluorescence signals should be absent in knockout samples)

• Peptide competition assays (pre-incubation with immunizing peptide should abolish specific signals)

• Cross-reactivity testing across species (most RASSF9 antibodies show reactivity with human, mouse, rat, and bovine proteins, with varying percentages of cross-reactivity)

The specificity of anti-RASSF9 antiserum has been confirmed by Western immunoblotting of exogenously expressed RASSF9 protein, demonstrating appropriate molecular weight detection .

What are the expected expression patterns of RASSF9 in normal tissues?

RASSF9 shows tissue-specific expression patterns that should be considered when selecting positive controls:

Quantification of RASSF9 immunofluorescent intensity using ImageJ software has revealed more prominent expression in suprabasal layers compared to basal and granular layers in normal skin . In situ hybridization with antisense probes against RASSF9 mRNA further confirms strong expression in the suprabasal layer of normal mouse epidermis .

What are the recommended protocols for Western blotting using RASSF9 antibodies?

For optimal Western blot results with RASSF9 antibodies:

• Use protein extraction buffers containing protease inhibitors to prevent degradation

• Load 20-40 μg of total protein per lane

• Recommended antibody concentration: 0.04-0.4 μg/mL

• Include positive controls (recombinant RASSF9 or tissues with known expression)

• Expected molecular weight: approximately 50 kDa (435 amino acid residues)

• Use 4-12% gradient gels for optimal separation

• Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

• Block with 5% non-fat milk or BSA in TBST

When comparing RASSF9 expression levels between samples, normalization to housekeeping proteins (β-actin, GAPDH) is essential for accurate quantification.

How should researchers design experiments to investigate RASSF9's role in the MEK/ERK signaling pathway?

To study RASSF9's role in MEK/ERK signaling, consider this experimental workflow:

• Overexpression and knockdown studies:

  • Transfect cells with plasmids carrying RASSF9 or siRNA targeting RASSF9

  • Confirm expression changes via Western blot

• Activation analysis:

  • Measure phosphorylation levels of MEK (p-MEK) and ERK (p-ERK) using phospho-specific antibodies

  • RASSF9 overexpression robustly stimulates p-ERK in NSCLC cell lines

• Pharmacological intervention:

  • Use MEK inhibitors (selumetinib, U0126) to block RASSF9-induced MEK/ERK activation

  • Analyze downstream effects on cell proliferation and viability

  • Include appropriate controls (vehicle-treated cells)

• Functional readouts:

  • Proliferation assays (EdU incorporation, cell viability)

  • Cell cycle analysis (cyclins expression)

  • Expression analysis of downstream targets (c-Myc, Pax, Fos)

Research has shown that RASSF9-stimulated p-ERK can be completely counteracted by MEK inhibitors in NSCLC cell lines, confirming pathway specificity .

What methodologies are recommended for studying RASSF9 protein-protein interactions?

To investigate RASSF9 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use anti-RASSF9 antibody for pull-down (2-5 μg per reaction)

  • Pre-clear lysates to reduce non-specific binding

  • Include negative controls (IgG, irrelevant antibody)

  • Elute with gentle conditions to maintain interactions

  • Analyze by Western blot for potential binding partners

• Proximity Ligation Assay (PLA):

  • Use antibodies against RASSF9 and suspected interaction partners

  • Allows visualization of protein interactions in situ with subcellular resolution

  • Quantify interaction signals using appropriate imaging software

• Recombinant protein approaches:

  • Express GST-RASSF9 fusion proteins as described in the literature:

    • Clone 285-1625nt fragment of mouse RASSF9 cDNA into pGEX-4T-1 vector

    • Transform into E. coli BL21 competent cells

    • Induce with 1 mM IPTG for 4 hr at 37°C

  • Use for pull-down assays with cell lysates

• Yeast two-hybrid screening:

  • Use RASSF9 as bait to identify novel interaction partners

  • Validate identified interactions using Co-IP or PLA

How can researchers effectively distinguish between RASSF9's contrasting roles in different tissues?

RASSF9 shows context-dependent functions, acting as a tumor suppressor in some tissues while promoting proliferation in others:

• Comparative expression analysis:

  • Use matched normal/tumor tissues

  • Quantify RASSF9 expression by qRT-PCR and Western blot

  • Compare with markers of proliferation or differentiation

• Functional studies in multiple cell types:

  • Implement consistent RASSF9 modulation (overexpression/knockdown) across different cell types

  • In keratinocytes: analyze differentiation markers and p21Cip1 expression

  • In cancer cells: measure proliferation markers and MEK/ERK activation

• Rescue experiments:

  • In RASSF9-deficient keratinocytes, re-introducing RASSF9 using recombinant adenovirus (Adv/HA-RASSF9) rescues terminal differentiation markers

  • Design similar rescue experiments for cancer cells to confirm specificity

• In vivo models:

  • Compare RASSF9-null mice phenotypes (growth retardation, short lifespan, less subcutaneous adipose layer, alopecia) with xenograft models showing RASSF9-promoted tumor growth

This dual nature of RASSF9 highlights the importance of cellular context in determining protein function.

What are the current technical challenges in developing high-specificity RASSF9 antibodies?

Developing highly specific RASSF9 antibodies faces several challenges:

• Epitope selection considerations:

  • The RASSF9 protein shares structural similarities with other RASSF family members

  • Optimal epitope regions include C-terminal sequences that show higher uniqueness

  • Immunogens often target sequences like YRILIDKLSAEIEKEVKSVCIDINEDAEGEAASELESSNLESVKCDLEKSMKAGLKIHSHLSGIQKEIKYSDSLLQMKAKEY

• Validation requirements:

  • Multi-method validation is essential (Western blot, IHC, IF, peptide blocking)

  • Cross-reactivity testing across RASSF family members

  • Testing across multiple species (human, mouse, rat, bovine)

  • Knockout/knockdown validation

• Application-specific optimization:

  • Different applications require different antibody formats

  • For immunofluorescence studies in epidermal tissues, antibodies must penetrate complex tissue architecture

  • For co-IP studies, antibodies must maintain high affinity under native conditions

• Recombinant antibody alternatives:

  • Moving beyond hybridoma technology to recombinant approaches

  • CRISPR/Cas9 genomic editing allows site-specific modifications to antibodies

  • Sortase tags can be incorporated for site-specific conjugation without impairing antigen binding

What methods are recommended for quantifying RASSF9 expression levels in experimental models?

For accurate quantification of RASSF9 expression:

• qRT-PCR:

  • Design primers specific to RASSF9 (avoiding cross-amplification with other RASSF family members)

  • Use multiple reference genes for normalization

  • Perform melting curve analysis to ensure specificity

• Protein quantification approaches:

  • Western blot with internal loading controls

  • ELISA development using validated antibodies

  • Reverse phase protein arrays (RPPA) for high-throughput analysis

• Image-based quantification:

  • For immunofluorescence: use ImageJ software for intensity measurement across tissue layers

  • For in situ hybridization: quantify signal intensity with appropriate controls

  • For IHC: use H-score or other semi-quantitative scoring systems

• Advanced techniques:

  • Mass spectrometry-based quantification

  • Immuno-MRM (Multiple Reaction Monitoring) for absolute quantification

When comparing normal and pathological samples, matching exposure settings and processing conditions is critical for valid comparisons.

How should researchers approach studying post-translational modifications of RASSF9?

Investigating post-translational modifications (PTMs) of RASSF9 requires:

• Phosphorylation analysis:

  • Immunoprecipitate RASSF9 and probe with anti-phospho antibodies

  • Use phosphatase inhibitors during protein extraction

  • Consider phospho-enrichment techniques before mass spectrometry

  • Investigate potential phosphorylation in relation to MEK/ERK pathway activation

• Other potential PTMs:

  • Ubiquitination (potential regulation of protein stability)

  • SUMOylation (potential regulation of protein-protein interactions)

  • Use specific inhibitors to block PTM pathways and observe effects on RASSF9 function

• Site-directed mutagenesis:

  • Identify potential PTM sites through bioinformatic prediction

  • Create site-specific mutants (e.g., phosphomimetic or phospho-deficient)

  • Test functional consequences in appropriate cellular models

• PTM-specific antibodies:

  • Development of modification-specific antibodies

  • Validate using both wild-type and mutant RASSF9 proteins

  • Use for spatio-temporal analysis of RASSF9 modifications