RASSF2 Antibody

What is the basic structure of RASSF2 protein and how does it influence antibody selection?

RASSF2 is a 34-36 kDa member of the RASSF family of proteins, comprising 326 amino acids in human form. The protein contains three key functional domains that are important when selecting antibodies for specific research applications :

• Bipartite nuclear localization signal (NLS) at amino acids 151-167

• Ras-association (RA) domain at amino acids 176-264

• SARAH domain at amino acids 272-319 that mediates homo- and heterotypic interactions

When selecting antibodies, researchers should consider which domain they wish to target based on their experimental goals. For instance, antibodies targeting the RA domain may be useful for studying RASSF2-Ras interactions, while those targeting the SARAH domain might be better for investigating interactions with other proteins like MST1/2.

How does RASSF2 function as a tumor suppressor, and which antibodies best detect its functional state?

RASSF2 acts as a tumor suppressor through several mechanisms:

• Binds directly to K-Ras in a GTP-dependent manner via its RA domain

• Promotes apoptosis and cell cycle arrest

• Stabilizes STK3/MST2 by protecting it from proteasomal degradation

• Suppresses NF-κB signaling

For studying the tumor suppressor function, antibodies that can detect the active, non-modified form of RASSF2 are ideal. Polyclonal antibodies that recognize multiple epitopes may be more effective for detecting functionally active RASSF2 in tumor samples where post-translational modifications might be altered.

What are the optimal applications for RASSF2 antibodies in cancer research?

Based on the research findings, RASSF2 antibodies have been successfully employed in various applications for cancer research:

For cancer research specifically, western blotting can effectively demonstrate RASSF2 downregulation in tumor samples, while immunohistochemistry can reveal its subcellular localization and expression patterns in tissue sections.

How should researchers optimize western blotting protocols for detecting RASSF2 in different cell lines?

Optimizing western blotting for RASSF2 detection requires attention to several critical factors:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent RASSF2 degradation

• Loading controls: β-actin is commonly used with RASSF2 detection

• Membrane selection: PVDF membranes have shown good results for RASSF2 detection

• Blocking solutions: 5% non-fat milk in TBST or BSA-based blockers may be used

• Primary antibody incubation: Typically overnight at 4°C with appropriate dilution

• Detection system: HRP-conjugated secondary antibodies with appropriate specificity

When working with different cell lines, it's important to note that RASSF2 appears at approximately 40 kDa in Jurkat, Raji, CEM, and Daudi human lymphoma cell lines as detected using Goat Anti-Human/Mouse RASSF2 antibodies . The experiment should be conducted under reducing conditions using appropriate immunoblot buffer systems.

What are common causes of non-specific binding with RASSF2 antibodies and how can they be mitigated?

Non-specific binding is a common issue with RASSF2 antibodies that can complicate data interpretation. Several strategies can address this problem:

• Antibody validation: Use knockout or knockdown models as negative controls

• Blocking optimization: Extend blocking time or adjust blocker composition

• Antibody dilution: Test a range of antibody dilutions to find optimal signal-to-noise ratio

• Wash stringency: Increase the number or duration of washing steps

• Use of detergents: Adjust Tween-20 concentration in wash buffers

• Pre-adsorption: Pre-adsorb antibodies with non-specific proteins

The presence of multiple RASSF2 isoforms can also lead to unexpected banding patterns. Research has identified at least three isoforms: one with a 13 amino acid insertion after Lys213, another with a three amino acid substitution for residues 305-326, and a third with a 20 amino acid substitution for residues 1-96 along with a three amino acid substitution for residues 231-326 . Researchers should be aware of these variants when interpreting western blot results.

How can researchers effectively distinguish between RASSF family members in experimental systems?

Distinguishing between RASSF family members is crucial for accurate data interpretation due to their structural similarities. Recommended approaches include:

• Antibody selection: Choose antibodies raised against unique regions of RASSF2 that do not share homology with other RASSF proteins

• Immunogen information: Verify the immunogen used for antibody production; for example, the OAAB22034 antibody was generated using a KLH-conjugated synthetic peptide from amino acids 123-156 of human RASSF2

• Validation experiments:

  • Overexpression systems with tagged RASSF proteins

  • siRNA knockdown of specific RASSF family members

  • Use of knockout cell lines or tissues

• Multiple detection methods: Combine different techniques (e.g., western blot and immunofluorescence)

• Subcellular localization analysis: RASSF2 is primarily nuclear, while other family members like RASSF1A are predominantly cytoplasmic

How can RASSF2 antibodies be employed to study its role in the Hippo signaling pathway?

RASSF2 has been implicated in the Hippo signaling pathway through its interaction with MST1/2. Researchers can investigate this relationship using these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use RASSF2 antibodies to pull down protein complexes and probe for MST1/2 and other Hippo pathway components

• Proximity ligation assays: Detect protein-protein interactions between RASSF2 and Hippo pathway components in situ

• Immunofluorescence co-localization: Determine subcellular co-localization of RASSF2 with MST1/2 using dual staining approaches

• Functional assays: Combine RASSF2 antibody detection with phosphorylation state analysis of downstream Hippo effectors like YAP/TAZ

• ChIP assays: Investigate whether RASSF2 associates with transcriptional complexes that regulate Hippo target genes

Research findings indicate that RASSF2 associates with and stabilizes MST1 and MST2 via the SARAH domain . This interaction suggests that RASSF2 may regulate organ size and cell proliferation through the Hippo pathway, making it an important area for further investigation.

What methodologies can be used to study RASSF2's role in bone remodeling and haematopoiesis?

RASSF2 knockout mice exhibit bone defects and haematopoietic anomalies, suggesting important roles in these processes. Researchers can investigate these functions using:

• Histological analysis: Use RASSF2 antibodies for immunohistochemistry of bone sections to detect expression patterns in osteoblasts and osteoclasts

• In vitro differentiation assays:

  • Osteoblast differentiation from mesenchymal stem cells

  • Osteoclast differentiation from hematopoietic precursors

  • Monitor RASSF2 expression throughout differentiation using western blotting and immunofluorescence

• Signaling studies:

  • Assess NF-κB activation states using phospho-specific antibodies alongside RASSF2 detection

  • Co-IP assays to detect RASSF2 interactions with IKKα and IKKβ

• Bone marrow transplantation experiments: Combine with RASSF2 antibody-based detection to track cell differentiation and lineage commitment

• RASSF2 rescue experiments: Reintroduce RASSF2 in knockout systems and monitor bone formation and haematopoiesis

Research has demonstrated that RASSF2 regulates osteoblast and osteoclast differentiation by inhibiting NF-κB signaling. In vitro studies showed that RASSF2 directly associates with IKKα and IKKβ and suppresses their activity .

What are the critical factors for successful immunofluorescence and immunohistochemistry with RASSF2 antibodies?

Successful immunostaining for RASSF2 requires attention to several technical parameters:

• Fixation methods: RASSF2 detection has been successful with immersion-fixed samples

• Antigen retrieval: May be necessary for formalin-fixed, paraffin-embedded samples

• Antibody concentration: 5 μg/mL has been effective for immunofluorescence in U937 cells

• Incubation conditions: 3 hours at room temperature has been validated for immunofluorescence

• Secondary antibody selection: Anti-species antibodies conjugated to appropriate fluorophores

• Counterstaining: DAPI is commonly used to visualize nuclei alongside RASSF2 staining

• Controls: Include positive and negative controls to validate staining specificity

For subcellular localization studies, it's important to note that RASSF2 has been detected in both the cytoplasm and nuclei of U937 human histiocytic lymphoma cells , though other studies report predominantly nuclear localization in certain cell types .

How do different fixation and permeabilization methods affect RASSF2 epitope accessibility?

The choice of fixation and permeabilization methods can significantly impact RASSF2 epitope accessibility and antibody binding:

• Aldehyde fixatives (formaldehyde, glutaraldehyde):

  • May mask epitopes through protein cross-linking

  • May require antigen retrieval methods (heat or enzymatic)

  • Preserve cellular architecture well but can reduce antibody penetration

• Organic solvent fixatives (methanol, acetone):

  • Generally better for preserving antigenic sites

  • May cause protein denaturation affecting conformational epitopes

  • Provide better permeabilization but can disrupt membrane structures

• Combined approaches:

  • Brief formaldehyde fixation followed by methanol permeabilization

  • Can balance structural preservation with epitope accessibility

For nuclear proteins like RASSF2, ensuring adequate nuclear permeabilization is crucial. Extended permeabilization times or stronger detergents (0.2-0.5% Triton X-100) may be necessary for optimal nuclear staining. Researchers should empirically determine the optimal fixation and permeabilization conditions for their specific RASSF2 antibody and cell type.

How can researchers investigate RASSF2's interaction with PAR-4 in prostate cancer research?

RASSF2 has been identified as an interactor with prostate apoptosis response protein 4 (PAR-4), a key tumor suppressor in prostate cancer . To study this interaction:

• Co-immunoprecipitation assays:

  • Immunoprecipitate RASSF2 from prostate cancer cell lysates and probe for PAR-4

  • Perform the reverse experiment by immunoprecipitating PAR-4 and probing for RASSF2

  • Use appropriate controls including IgG controls and input samples

• Proximity ligation assay (PLA):

  • Visualize and quantify RASSF2-PAR-4 interactions in situ

  • Compare interaction levels between normal and cancerous prostate tissues

• FRET or BRET analysis:

  • Generate fluorescent or bioluminescent tagged versions of RASSF2 and PAR-4

  • Measure energy transfer as an indicator of protein-protein interaction

• Domain mapping:

  • Generate truncated versions of RASSF2 to identify which domains interact with PAR-4

  • Use antibodies specific to different RASSF2 domains in co-IP experiments

• Functional studies:

  • Assess how RASSF2 knockdown affects PAR-4-mediated apoptosis

  • Investigate whether this interaction affects K-Ras signaling

This interaction may provide insights into RASSF2's role as a tumor suppressor in prostate cancer and could potentially be exploited for therapeutic approaches.

What techniques are most effective for studying RASSF2's role in NF-κB signaling regulation?

RASSF2 has been shown to regulate NF-κB signaling by interacting with IKK complexes. Researchers can investigate this mechanism using:

• NF-κB reporter assays:

  • Use firefly luciferase reporter plasmid pNF-κB-Luc alongside Renilla luciferase control

  • Compare NF-κB activity in cells with normal versus altered RASSF2 expression

• IKK kinase activity assays:

  • In vitro kinase assays using GST-IKKβ, GST-IκBα, and purified RASSF2 protein

  • Measure phosphorylation of IκBα by autoradiography in the presence of different molar ratios of RASSF2

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions to assess NF-κB translocation

  • Use NE-PER Nuclear and Cytoplasmic Extraction Reagents

• Immunostaining for phospho-p65:

  • Detect NF-κB activation status by immunofluorescence

  • Compare nuclear translocation patterns in control versus RASSF2-deficient cells

• Western blot analysis of pathway components:

  • Monitor phosphorylation status of IκBα

  • Track degradation of IκBα in response to stimuli

  • Assess nuclear accumulation of p65

Research has demonstrated that RASSF2 associates with both IKKα and IKKβ forms and suppresses IKK activity. Introduction of either RASSF2 or a dominant-negative form of IKK into Rassf2-/- cells inhibited NF-κB hyperactivation and normalized cellular differentiation .

How can RASSF2 antibodies be utilized in comparative studies across different species models?

RASSF2 is conserved across species, allowing for comparative studies using appropriate antibodies:

• Antibody selection for cross-species studies:

  • Choose antibodies raised against conserved epitopes

  • Verify cross-reactivity with the target species

  • RASSF2 Antibody (E-11) detects RASSF2 protein in mouse, rat, and human samples

  • Human/Mouse RASSF2 Antibody (AF5639) specifically recognizes both human and mouse RASSF2

• Western blot optimization:

  • Adjust protein loading amounts based on expression levels in different species

  • Modify transfer conditions for proteins from different species

  • Use species-appropriate positive controls

• Immunohistochemistry across species:

  • Optimize antigen retrieval methods for each species' tissue

  • Adjust antibody concentrations based on cross-reactivity efficiency

  • Use species-specific blocking reagents to reduce background

• Knockout/knockdown validation:

  • Use RASSF2 knockout mice tissues as negative controls

  • Compare staining patterns between wildtype and knockout samples across species

• Evolutionary studies:

  • Analyze RASSF2 expression patterns across evolutionary distant species

  • Correlate structural conservation with functional conservation

These approaches can provide insights into the evolutionary conservation of RASSF2 function and its role in different model organisms.

What are the best practices for using RASSF2 antibodies in studies of epigenetic silencing through promoter hypermethylation?

RASSF2 is frequently inactivated in tumors via promoter hypermethylation. To study this epigenetic regulation:

• Combined methylation and expression analysis:

  • Perform bisulfite sequencing or methylation-specific PCR of the RASSF2 promoter

  • Correlate with protein expression levels using RASSF2 antibodies in the same samples

  • Compare expression in normal versus tumor tissues

• Demethylation studies:

  • Treat cells with demethylating agents like 5-aza-2'-deoxycytidine

  • Monitor RASSF2 re-expression using antibodies in western blot or immunofluorescence

  • Assess restoration of tumor suppressor function

• ChIP-based approaches:

  • Use antibodies against histone modifications associated with silenced chromatin

  • Correlate histone modification patterns with RASSF2 expression levels

  • Perform sequential ChIP to analyze complex epigenetic patterns

• Correlation studies in clinical samples:

  • Analyze RASSF2 promoter methylation in tumor samples

  • Use immunohistochemistry with RASSF2 antibodies on the same samples

  • Correlate methylation status with protein expression and clinical outcomes

• Functional rescue experiments:

  • Reintroduce RASSF2 in cells with hypermethylated promoters

  • Use antibodies to confirm expression and assess restoration of tumor suppressor functions

RASSF2 has been shown to be inactivated in various tumors including colorectal cancer cells via CpG island promoter hypermethylation , making this an important area for cancer research.

How can researchers leverage RASSF2 antibodies to explore its potential as a biomarker or therapeutic target?

RASSF2's role as a tumor suppressor presents opportunities for biomarker development and therapeutic targeting:

• Biomarker development approaches:

  • Analyze RASSF2 expression in tissue microarrays from various cancer types

  • Correlate expression levels with clinical outcomes and treatment responses

  • Develop standardized immunohistochemistry scoring systems

• Early detection strategies:

  • Assess RASSF2 expression in precancerous lesions

  • Correlate with progression to invasive cancer

  • Combine with other biomarkers for improved sensitivity and specificity

• Therapeutic target validation:

  • Screen for compounds that can restore RASSF2 expression in hypermethylated cells

  • Develop peptide mimetics that can recapitulate RASSF2's interaction with K-Ras

  • Create cell-penetrating constructs that can deliver functional RASSF2 protein domains

• Combination therapy approaches:

  • Investigate how RASSF2 restoration might sensitize cells to conventional therapies

  • Study potential synergies with inhibitors of the Ras pathway or NF-κB signaling

• Immunotherapy implications:

  • Explore whether RASSF2 expression correlates with immunotherapy response

  • Investigate potential interactions between RASSF2 and immune checkpoint molecules

Given RASSF2's interactions with the Ras signaling pathway, which is essential for transmitting signals from cell surface receptors to various intracellular effectors , targeting this interaction could provide novel therapeutic opportunities.

What novel techniques might enhance RASSF2 detection and functional characterization in complex biological samples?

Emerging technologies offer new opportunities for studying RASSF2:

• Mass spectrometry-based approaches:

  • Targeted proteomics to quantify RASSF2 protein levels

  • Phosphoproteomics to identify post-translational modifications

  • Interaction proteomics to discover novel RASSF2 binding partners

• High-resolution microscopy techniques:

  • Super-resolution microscopy to visualize RASSF2 localization at nanoscale resolution

  • Live-cell imaging with fluorescently tagged RASSF2 to track dynamic interactions

  • FRET-based biosensors to monitor RASSF2 activity in real-time

• Single-cell technologies:

  • Single-cell proteomics to analyze RASSF2 expression heterogeneity within tumors

  • Combine with transcriptomics for multi-omic profiling

  • Spatial proteomics to map RASSF2 expression in the tissue microenvironment

• CRISPR-based studies:

  • CRISPR activation to upregulate endogenous RASSF2

  • Domain-specific mutagenesis to dissect functional roles

  • CRISPR screens to identify synthetic lethal interactions

• Computational approaches:

  • Machine learning algorithms to predict RASSF2 functionality from sequence or structure

  • Network analysis to place RASSF2 in broader signaling contexts

  • Virtual screening for compounds that might modulate RASSF2 function