RASSF2 Antibody, Biotin conjugated

What are the primary cellular functions of RASSF2?

RASSF2 functions as a potential tumor suppressor through multiple mechanisms. It serves as a KRAS-specific effector protein that promotes apoptosis and cell cycle arrest . RASSF2 stabilizes STK3/MST2 by protecting it from proteasomal degradation and may participate in the Hippo signaling pathway . Recent research has uncovered RASSF2's critical role in bone remodeling, where it regulates both osteoblast and osteoclast differentiation by inhibiting NF-κB signaling through direct interaction with IκB kinase (IKK) α and β . This mechanistic pathway is evidenced by co-immunoprecipitation experiments demonstrating that RASSF2 associates with IKKα and IKKβ, subsequently inhibiting IKK activity and affecting downstream NF-κB signaling cascades .

Why are biotin-conjugated antibodies useful for RASSF2 research?

Biotin-conjugated RASSF2 antibodies provide significant advantages for detection sensitivity and experimental flexibility. The biotin-streptavidin system offers one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), enabling robust signal amplification in various detection methods . This conjugation allows researchers to leverage multiple secondary detection systems, including streptavidin-HRP, streptavidin-fluorophores, or streptavidin-gold nanoparticles, without changing the primary antibody . Additionally, biotin conjugation enables multi-parameter experiments where several antigens can be detected simultaneously using different visualization systems, particularly valuable for co-localization studies examining RASSF2 interaction with signaling partners like IKK complexes .

How can biotin-conjugated RASSF2 antibodies be optimally used in immunofluorescence microscopy?

For optimal immunofluorescence microscopy using biotin-conjugated RASSF2 antibodies, implement the following protocol:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 10% goat serum for 1 hour at room temperature

• Incubate with biotin-conjugated RASSF2 antibody (5 μg/mL) overnight at 4°C

• Detect using streptavidin-conjugated fluorophores (e.g., NorthernLights™ 557-conjugated streptavidin)

• Counterstain nuclei with DAPI

This approach has been validated for detecting RASSF2 in multiple cell types, including U937 human histiocytic lymphoma cells, where specific staining was localized to both cytoplasm and nuclei . When designing co-localization experiments, consider that RASSF2 has been shown to interact with IKK complexes, suggesting examination of both nuclear and cytoplasmic compartments .

What are the recommended approaches for detecting RASSF2 in flow cytometry using biotin-conjugated antibodies?

For flow cytometric detection of RASSF2 using biotin-conjugated antibodies, the following methodology is recommended:

• Fix cells with 4% paraformaldehyde to facilitate intracellular staining

• Permeabilize cells with appropriate permeabilization buffer

• Block with 10% normal goat serum to minimize non-specific binding

• Incubate with biotin-conjugated RASSF2 antibody (1 μg per 1×10^6 cells) for 30 minutes at 20°C

• Detect using streptavidin-conjugated fluorophores (e.g., DyLight®488)

• Include appropriate controls:

  • Isotype control antibody (e.g., rabbit IgG at 1 μg per 1×10^6 cells)

  • Unstained control (cells without primary and secondary reagents)

This protocol has been validated in HL-60 cells . For multiparameter analysis, RASSF2 detection can be combined with markers of apoptosis or cell cycle, relevant to RASSF2's known functions in apoptosis promotion and cell cycle regulation .

What are the validated applications for biotin-conjugated RASSF2 antibodies in current research?

Biotin-conjugated RASSF2 antibodies have been validated for multiple research applications:

For each application, antibody concentration should be optimized based on experimental conditions and target cell types. Expression levels of RASSF2 can vary significantly across different tissues and cell lines .

How does RASSF2 modulate NF-κB signaling in osteoblast and osteoclast differentiation?

RASSF2 functions as a critical negative regulator of NF-κB signaling through direct inhibition of IKK activity, with significant implications for bone remodeling. Biochemical studies have demonstrated that RASSF2 physically associates with both IKKα and IKKβ through co-immunoprecipitation experiments . In vitro kinase assays using purified RASSF2, IKKβ, and IκBα proteins have conclusively shown that RASSF2 prevents IKKβ-mediated IκBα phosphorylation in a dose-dependent manner .

In osteoclast precursors, RASSF2 deletion results in enhanced RANKL-induced NF-κB activation, promoting osteoclastogenesis through increased expression of osteoclast markers like Acp5, Ctsk, Oscar, c-Src, c-Fos, and NFATc1 . Conversely, in osteoblast precursors, RASSF2 deficiency leads to constitutive NF-κB activation that suppresses osteoblastogenesis .

Genetic complementation experiments have confirmed this mechanism, as reintroduction of RASSF2 into Rassf2-/- cells normalizes NF-κB signaling and restores both osteoclast and osteoblast differentiation to wild-type levels . Similarly, expression of dominant-negative IKKγ in Rassf2-/- cells rescues the differentiation phenotypes, demonstrating that RASSF2's effects on bone cell differentiation are primarily mediated through IKK inhibition .

What is the role of RASSF2 in colorectal cancer and how can biotin-conjugated antibodies help investigate this function?

RASSF2 acts as a tumor suppressor in colorectal cancer (CRC) through regulation of Ras signaling. Research has established that RASSF2 is frequently silenced in CRC through aberrant methylation and histone deacetylation . Functional studies have demonstrated RASSF2's tumor-suppressive activities, including induction of morphological changes, promotion of apoptosis, and prevention of cell transformation .

Biotin-conjugated RASSF2 antibodies can effectively investigate this function through:

• Chromatin immunoprecipitation (ChIP) assays to examine RASSF2 promoter methylation status and associated histone modifications

• Immunofluorescence studies to analyze RASSF2 subcellular localization changes in response to oncogenic K-ras signaling

• Flow cytometric analysis to quantify apoptosis induction by RASSF2 in CRC cell lines

• Co-immunoprecipitation studies to identify RASSF2 interaction partners in the Ras signaling pathway

Particularly significant is the finding that primary CRCs with K-ras/BRAF mutations frequently display RASSF2 methylation, and RASSF2 inactivation enhances K-ras-induced oncogenic transformation . This suggests a model where RASSF2 normally functions as a negative regulator of oncogenic Ras signaling, and its silencing contributes to colorectal tumorigenesis .

How do biotin-conjugated RASSF2 antibodies overcome the uptake limitations of biotin conjugates?

Several mechanisms may explain how biotin-conjugated RASSF2 antibodies overcome this limitation:

• Alternative recognition pathways: Biotin conjugates may utilize distinct "biotin receptors" rather than transporters, though such receptors have not been conclusively identified

• Conformational adaptations: The conjugated biotin may adopt conformations that partially mimic the recognition features of free biotin

• Non-SMVT transport systems: Evidence suggests the existence of alternative biotin transport systems in specific cell types. For instance, human keratinocytes possess a second uptake system for biotin with a Michaelis-Menten constant of 2.6 nM that is not inhibited by lipoic acid or pantothenic acid, unlike SMVT

• Endocytosis-mediated uptake: Biotin-conjugated antibodies may enter cells through conventional antibody internalization pathways rather than biotin-specific transport

Understanding these mechanisms is critical for experimental design, particularly for applications requiring cellular internalization of biotin-conjugated RASSF2 antibodies .

How can researchers address non-specific binding issues with biotin-conjugated RASSF2 antibodies?

Non-specific binding is a common challenge with biotin-conjugated antibodies due to endogenous biotin present in many biological samples. To minimize this issue with RASSF2 detection:

• Implement a biotin blocking step:

  • Pre-treat samples with avidin followed by biotin (sequential blocking)

  • Alternatively, use commercial biotin blocking kits specifically designed for immunohistochemistry/immunofluorescence

• Validate antibody specificity:

  • Use RASSF2 knockout or knockdown cells as negative controls

  • Compare staining patterns with multiple RASSF2 antibodies recognizing different epitopes

  • Perform peptide competition assays

• Optimize blocking conditions:

  • Extend blocking time with 10% normal goat serum to 1-2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to blocking solution for intracellular staining

  • Consider adding 1% BSA to reduce background

• Use appropriate controls:

  • Include isotype controls at the same concentration as the primary antibody

  • Use secondary-only controls to identify background from the detection system

These approaches have been validated across multiple experimental systems including immunocytochemistry of U2OS cells and flow cytometry of HL-60 cells with biotin-conjugated RASSF2 antibodies .

What are the optimal conditions for detecting RASSF2-IKK interactions using biotin-conjugated antibodies?

For optimal detection of RASSF2-IKK interactions using biotin-conjugated antibodies, consider the following validated methodology:

• Cell lysis optimization:

  • Use a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, and 5 mM EDTA

  • Include protease inhibitors (PMSF, aprotinin, leupeptin) and phosphatase inhibitors

  • Maintain low temperature (4°C) throughout the procedure

• Immunoprecipitation protocol:

  • Pre-clear lysates with protein A/G beads for 1 hour

  • Incubate pre-cleared lysates with biotin-conjugated RASSF2 antibody (5 μg per 1 mg of total protein) overnight at 4°C

  • Capture complexes using streptavidin-conjugated magnetic beads

  • Wash extensively (4-5 times) with lysis buffer containing reduced detergent (0.1% NP-40)

• Detection strategies:

  • Analyze precipitates by western blotting for IKKα and IKKβ

  • Consider reverse co-IP with IKK antibodies to confirm interaction

  • Evaluate interaction under various stimulation conditions (e.g., TNFα treatment)

• Functional validation:

  • Perform in vitro kinase assays using immunoprecipitated complexes with GST-IκBα as substrate

  • Compare kinase activity in the presence and absence of purified RASSF2

This approach has successfully demonstrated that RASSF2 associates with both IKKα and IKKβ and inhibits their kinase activity, establishing RASSF2 as a negative regulator of NF-κB signaling .

What methodological adjustments are needed for analyzing RASSF2 expression in bone tissue samples?

Analyzing RASSF2 expression in bone tissue presents unique challenges requiring specific methodological adjustments when using biotin-conjugated antibodies:

• Sample preparation considerations:

  • For paraffin-embedded sections, use EDTA-based decalcification rather than acid-based methods to preserve antigenicity

  • For frozen sections, employ cryofilm techniques to maintain tissue integrity

  • Consider thickness of 5-7 μm for optimal antibody penetration

• Antigen retrieval optimization:

  • Use enzyme-based antigen retrieval (demonstrated effective for RASSF2 detection)

  • Extend retrieval time to 15-20 minutes for bone tissues

  • Alternatively, try heat-mediated retrieval in citrate buffer (pH 6.0)

• Detection system modifications:

  • Employ streptavidin-polymer based detection systems for enhanced sensitivity

  • For fluorescent detection, use longer incubation times (overnight at 4°C) with primary antibody

  • Counter-label with osteoblast markers (Runx2, Osterix) or osteoclast markers (TRAP, Cathepsin K) for contextual analysis

• Controls and quantification:

  • Include Rassf2-/- bone tissue as negative control when available

  • Quantify RASSF2 expression in relation to cell-specific markers

  • Analyze both trabecular and cortical bone regions separately

These adjustments have been validated in studies examining the role of RASSF2 in bone remodeling, where immunohistochemical analysis revealed decreased numbers of osteoblasts and osteoclasts in Rassf2-/- mice compared to wild-type controls .

How can biotin-conjugated RASSF2 antibodies help investigate the protein's role in the Hippo signaling pathway?

RASSF2 has been implicated in the Hippo signaling pathway, which regulates organ size and tissue homeostasis. Biotin-conjugated RASSF2 antibodies can facilitate investigation of this connection through:

• Proximity ligation assays (PLA) to detect in situ interactions between RASSF2 and Hippo pathway components like MST1/2 (mammalian STE20-like kinases)

• ChIP-seq experiments to identify RASSF2 binding sites on chromatin and potential co-regulation with YAP/TAZ transcription factors

• Immunofluorescence co-localization studies to analyze RASSF2 dynamics relative to Hippo pathway components during different cellular states

• Protein complex isolation through sequential immunoprecipitation using biotin-conjugated RASSF2 antibodies followed by mass spectrometry

Research has established that RASSF2 stabilizes STK3/MST2 by protecting it from proteasomal degradation , suggesting a mechanism by which RASSF2 might influence Hippo pathway activation. The development of methodologies using biotin-conjugated antibodies to track these interactions under different cellular conditions could provide critical insights into RASSF2's role in connecting Ras and Hippo signaling networks .

What methodological considerations are important when using biotin-conjugated RASSF2 antibodies to study its tumor suppressor function?

When investigating RASSF2's tumor suppressor function using biotin-conjugated antibodies, implement these methodological considerations:

• Cell line selection strategies:

  • Compare colorectal cancer cell lines with known RASSF2 methylation status

  • Include matched pairs of RASSF2-expressing and RASSF2-silenced cells

  • Consider cells with various K-ras mutation statuses to examine RASSF2-Ras interactions

• Expression restoration experiments:

  • Use inducible RASSF2 expression systems to monitor temporal effects

  • Analyze changes in cell morphology, apoptosis, and cell cycle upon RASSF2 restoration

  • Implement time-course immunofluorescence to track subcellular localization shifts

• Signaling pathway analysis:

  • Examine NF-κB pathway components before and after RASSF2 restoration

  • Investigate RASSF2's effect on K-ras-induced transformation

  • Monitor apoptosis markers in relation to RASSF2 expression levels

• In vivo tumor models:

  • Develop xenograft models with biotin-conjugated antibody-based imaging

  • Analyze tumor sections for RASSF2 expression in relation to proliferation and apoptosis markers

  • Compare growth rates between RASSF2-positive and RASSF2-negative tumors