RASSF2 Antibody, FITC conjugated

What is RASSF2 and what methodologies are best for studying its function?

RASSF2 belongs to the Ras-association domain family (RASSF) of proteins, which plays a significant role in the Hippo signaling pathway. It functions as a tumor suppressor and a K-Ras-specific effector protein that promotes apoptosis and cell cycle arrest . RASSF2 contains a Ras-associating domain and a SARAH domain, which are important for its protein interactions .

Research methodologies for studying RASSF2 function include:

• Gene knockout models: RASSF2-/- mice exhibit systematic phenotypes including haematopoietic anomalies and bone remodeling defects

• Protein-protein interaction studies: Co-immunoprecipitation assays have revealed RASSF2 interacts with IKKα and IKKβ, inhibiting NF-κB signaling

• Molecular signaling analysis: In vitro kinase assays have demonstrated that RASSF2 prevents IKKβ-mediated IκBα phosphorylation in a dose-dependent manner

RASSF2 is widely expressed with highest levels in brain, placenta, peripheral blood, and lung, but is frequently down-regulated in lung tumor cell lines through promoter hypermethylation .

What is the significance of FITC conjugation for antibodies in research applications?

FITC (Fluorescein Isothiocyanate) is a green fluorescent dye with an excitation wavelength of 495nm and emission wavelength of 519nm . Conjugating FITC to antibodies provides several research advantages:

• Enables direct visualization in fluorescence microscopy with minimal background

• Allows quantitative analysis through flow cytometry

• Facilitates multiplexing with other fluorophores in co-localization studies

The conjugation process involves the reaction of FITC with primary amines on antibodies under controlled conditions. Optimal FITC conjugation is achieved when:

• Using relatively pure IgG (obtained by DEAE Sephadex chromatography)

• High-quality FITC is employed

• Reaction conditions are controlled: maximal labeling is obtained in 30–60 minutes at room temperature, pH 9.5, and an initial protein concentration of 25 mg/ml

The molecular fluorescein/protein (F/P) ratio is critical, as separation of optimally labeled antibodies from under- and over-labeled proteins significantly impacts detection sensitivity .

How should researchers design experiments to study RASSF2 subcellular localization using FITC-conjugated antibodies?

When designing experiments to study RASSF2 subcellular localization using FITC-conjugated antibodies, researchers should consider:

Experimental Setup Parameters:

• RASSF2 alone is primarily nuclear, but colocalizes in the cytoplasm when co-expressed with MST1 or MST2

• Differential expression patterns may exist between cell types and cancer subtypes

Recommended Protocol:

• Fix cells with 4% paraformaldehyde

• Permeabilize with appropriate buffer (e.g., 0.1% Triton X-100)

• Block with 10% normal goat serum

• Incubate with FITC-conjugated RASSF2 antibody (5 μg/mL is typically effective)

• For co-localization studies, include cytoskeletal markers such as phalloidin or tubulin antibodies

• Counterstain nuclei with DAPI

• Visualize using appropriate filter sets for FITC (excitation: 495nm, emission: 519nm)

Control Considerations:

• Include isotype controls to assess non-specific binding

• Use RASSF2-knockdown or knockout cells as negative controls

• Consider demethylating agent treatment (5-azadC and TSA) in cells with suppressed RASSF2 expression, as this has been shown to significantly increase RASSF2 protein expression in cytoplasmic and nuclear locations (p < 0.001)

What molecular mechanisms underlie RASSF2's role in bone remodeling, and how can FITC-conjugated antibodies help elucidate these pathways?

RASSF2 plays a crucial role in bone remodeling through regulation of osteoblast and osteoclast differentiation. FITC-conjugated RASSF2 antibodies can help visualize these interactions in cellular contexts.

Molecular Mechanisms:

• RASSF2 deficiency results in hyperactivation of NF-κB during both osteoclast and osteoblast differentiation

• RASSF2 associates with IKKα and IKKβ, suppressing IKK activity

• In vitro studies show that RASSF2 inhibits osteoclastogenesis but promotes osteoblastogenesis

• RASSF2-/- mice exhibit:

  • Systematic bone defects

  • Decreased numbers of osteoblasts and osteoclasts in bone

  • Enhanced formation of TRAP-positive multinucleated cells in vitro

  • Increased pit formation on dentine slices compared with wild-type cells

Research Approach Using FITC-Conjugated Antibodies:

• Use FITC-conjugated RASSF2 antibodies to track protein localization during osteoblast/osteoclast differentiation

• Perform time-course studies to visualize RASSF2 translocation during RANKL stimulation

• Combine with phospho-specific antibodies against IκBα and p65 to correlate RASSF2 localization with NF-κB activation status

• Implement dual staining with markers for osteoclast (TRAP, cathepsin K) or osteoblast (Runx2, osteocalcin) differentiation

The reintroduction of RASSF2 into RASSF2-/- cells normalizes NF-κB signaling and restores proper differentiation, providing a valuable experimental model for validation .

What are the optimal conditions for FITC conjugation to RASSF2 antibodies?

Achieving optimal FITC conjugation to RASSF2 antibodies requires careful control of reaction conditions to maximize fluorescence intensity while preserving antibody specificity and affinity.

Optimized Conjugation Protocol:

• Purify antibody using DEAE Sephadex chromatography to obtain high-quality IgG

• Adjust antibody to concentration of 10-25 mg/ml in an amine-free buffer

• Maintain pH between 7.3-7.6 (optimal: pH 9.5) using appropriate modifier reagent

• Add 1 µl of modifier reagent to each 10 µl of antibody solution

• Mix FITC with antibody and incubate at room temperature (20-25°C) for 3 hours in the dark

• Optional: Extend incubation overnight (longer times do not negatively affect conjugate)

Buffer Considerations:

• Buffers including MES, MOPS, HEPES, and phosphate are compatible at 10-50mM with pH 6.5-8.5

• Common non-buffering salts (e.g., sodium chloride), chelating agents (e.g., EDTA), and sugars may be present

• Sodium azide (0.02-0.1%) has little or no effect on the conjugation process

Critical Parameters Affecting Conjugation Efficiency:

How can researchers validate the specificity of FITC-conjugated RASSF2 antibodies?

Validating the specificity of FITC-conjugated RASSF2 antibodies is essential for accurate experimental results. Multiple complementary approaches should be employed:

Genetic Validation:

• Test antibody in RASSF2-knockout or knockdown models:

  • Use RASSF2−/− mice tissues or cells

  • Employ siRNA-mediated knockdown of RASSF2 expression

• Rescue experiments:

  • Reintroduce RASSF2 into RASSF2−/− cells using retroviral vectors (e.g., pMX-Flag–RASSF2)

  • Compare staining patterns before and after rescue

Biochemical Validation:

• Western blot analysis to confirm antibody recognizes a single band of the expected molecular weight (37-38 kDa)

• Peptide competition assays to demonstrate signal specificity

• Cross-reactivity testing against other RASSF family members (particularly RASSF1A, which shares structural similarities)

Flow Cytometry Validation:

• Include appropriate controls:

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

  • Unlabeled sample without primary and secondary antibodies

• Compare staining patterns between cell lines with known high RASSF2 expression (e.g., A549) and those with low expression due to promoter hypermethylation

What are the best methodologies for detecting RASSF2 protein interactions using FITC-labeled antibodies?

Investigating RASSF2 protein interactions is crucial to understanding its function. FITC-labeled antibodies offer several methodological advantages:

Co-immunoprecipitation Followed by Fluorescent Detection:

• Immunoprecipitate using RASSF2 antibody conjugated to Sepharose beads

• Detect interacting proteins using FITC-labeled antibodies against suspected binding partners

• For validation, perform reverse co-IP using antibodies against interacting proteins (e.g., MST1/2, IKKα/β)

The strongest validated RASSF2 binding partners include:

• MST1 and MST2 (identified by yeast two-hybrid screen and confirmed by mass spectrometry with 29.8% and 52.1% peptide coverage, respectively)

• IKKα and IKKβ (confirmed by endogenous co-immunoprecipitation)

• PAR-4 (identified as a direct binding partner in a two-hybrid screen)

Fluorescence Co-localization Studies:

• Use FITC-conjugated RASSF2 antibody alongside differently labeled antibodies against interacting proteins

• Analyze subcellular distribution patterns in different cell types

• Assess changes in localization following stimulation (e.g., RANKL treatment for NF-κB pathway activation)

FRET (Förster Resonance Energy Transfer):
For detecting direct protein-protein interactions:

• Label RASSF2 antibody with FITC (donor fluorophore)

• Label interacting protein antibody with a compatible acceptor fluorophore

• Measure energy transfer as evidence of close molecular proximity

How does RASSF2 expression vary across different cancer types, and what methodology is optimal for detecting these variations?

RASSF2 expression patterns vary significantly across cancer types, with important clinical implications. Multiple methodologies offer complementary approaches for detection:

Expression Patterns in Cancer:

• RASSF2 is frequently down-regulated in lung tumor cell lines

• In breast cancer, RASSF2 hypermethylation is significantly more frequent in luminal subtypes than non-luminal tumors (p = 0.001)

• RASSF2 hypermethylation is associated with better prognosis in multivariate statistical analysis (P < 0.05)

• Different immunohistochemical staining patterns (Pattern A and Pattern B) correlate with unmethylated and methylated status, respectively

Optimal Detection Methodologies:

• DNA Methylation Analysis:

  • Bisulfite sequencing or methylation-specific PCR to analyze RASSF2 promoter CpG islands

  • Particularly relevant as RASSF2 is inactivated in various tumors by promoter hypermethylation

• Immunohistochemistry:

  • Allows visualization of expression patterns in tissue context

  • Can distinguish different RASSF2 expression patterns (A and B) in tumors

  • Protocol: Use enzyme antigen retrieval (15 mins), block with 10% goat serum, incubate with RASSF2 antibody overnight at 4°C

• Immunofluorescence:

  • Higher sensitivity for detecting subcellular localization

  • Particularly valuable when coupled with demethylating agents (5-azadC and TSA) to assess re-expression

  • Quantification of nuclear vs. cytoplasmic expression provides additional insights

• Western Blotting:

  • Quantitative assessment of total protein levels

  • Can detect changes following treatment with demethylating agents

How can FITC-conjugated RASSF2 antibodies be used to study the RASSF2-MST1/2 tumor suppressor pathway?

The RASSF2-MST1/2 interaction represents a critical tumor suppressor pathway. FITC-conjugated antibodies provide valuable tools for investigating this system:

Pathway Overview:

• RASSF2 binds to and stabilizes the proapoptotic kinases MST1 and MST2

• RASSF2 protects MST2 from proteasomal degradation

• This interaction promotes apoptosis and cell cycle arrest

• RASSF2 and MST1/2 colocalize in the cytoplasm, although RASSF2 alone is nuclear

Research Methodologies:

• Domain Mapping Studies:

  • The interaction is mediated via the RASSF2 SARAH domain

  • RASSF2 constructs lacking the SARAH domain (amino acids 1-248) fail to interact with MST1/2

  • Full-length RASSF2 and RASSF2 Δ108-253 (lacking NLS and most of RA domain) maintain MST1/2 binding

• Phosphorylation Analysis:

  • RASSF2 can be phosphorylated by a co-immunoprecipitating kinase (likely MST1/2)

  • This phosphorylation does not induce RASSF2 dissociation from MST2

  • Use FITC-conjugated phospho-specific antibodies alongside RASSF2 antibodies to track phosphorylation status

• Live Cell Imaging:

  • Track dynamic interactions between RASSF2 and MST1/2 in response to apoptotic stimuli

  • Monitor subcellular relocalization events that may correlate with tumor suppressor activity

• Interaction Enhancement Studies:

  • The RASSF2-MST1/2 interaction can be enhanced in the presence of activated K-Ras

  • Use FITC-conjugated RASSF2 antibodies to quantify complex formation with and without Ras activation

Experimental Validation Controls:

• Use cells with RASSF2 knockdown to confirm antibody specificity

• Include appropriate isotype controls for fluorescence studies

• Consider co-staining with markers of apoptosis to correlate with pathway activation

What are the optimal protocols for detecting RASSF2 using FITC-conjugated antibodies in flow cytometry?

Flow cytometry with FITC-conjugated RASSF2 antibodies enables quantitative single-cell analysis of expression levels. The following protocol optimizes detection while minimizing background:

Optimized Protocol:

• Cell Preparation:

  • Harvest cells (e.g., HL-60) in logarithmic growth phase

  • Wash with PBS containing 1% BSA

  • Fix with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with appropriate permeabilization buffer

• Blocking and Staining:

  • Block with 10% normal goat serum (30 minutes at room temperature)

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

  • Wash three times with PBS/1% BSA

• Controls:

  • Isotype control: Use same concentration of FITC-conjugated rabbit IgG (1 μg per 1×10^6 cells)

  • Unstained control: Process cells without primary antibody

  • Positive control: Use cell line with known high RASSF2 expression (e.g., A549)

• Acquisition and Analysis:

  • Use appropriate voltage settings for FITC channel (488nm excitation)

  • Collect minimum of 10,000 events per sample

  • Analyze using histogram overlay to compare sample with controls

  • Quantify by measuring median fluorescence intensity (MFI)

Critical Parameters:

• Fixation time: Over-fixation can mask epitopes

• Antibody concentration: Titrate to determine optimal signal-to-noise ratio

• Incubation temperature: Room temperature (20°C) provides optimal binding kinetics

• Washing steps: Thorough washing reduces background fluorescence

How can researchers use FITC-conjugated RASSF2 antibodies to analyze tumor samples in translational research?

FITC-conjugated RASSF2 antibodies offer valuable tools for translational cancer research, enabling correlation between RASSF2 expression patterns and clinical outcomes:

Clinical Sample Processing:

• Fresh Tumor Samples:

  • Enzymatically dissociate tissue into single-cell suspension

  • Fix and permeabilize as described in flow cytometry protocol

  • Analyze RASSF2 expression alongside relevant markers (e.g., cell type, proliferation)

• FFPE Tissue Sections:

  • Perform antigen retrieval using enzyme antigen retrieval reagent (15 mins)

  • Block with 10% goat serum

  • Incubate with FITC-conjugated RASSF2 antibody (5 μg/mL)

  • Counterstain with DAPI

  • Analyze patterns (nuclear vs. cytoplasmic) and intensity

Translational Applications:

• Prognostic Marker Assessment:

  • RASSF2 hypermethylation is associated with better prognosis in certain cancers

  • Quantify RASSF2 expression in tumor samples and correlate with patient outcomes

  • Compare expression patterns between tumor subtypes (e.g., luminal vs. triple-negative breast cancer)

• Treatment Response Prediction:

  • Monitor RASSF2 expression before and after treatment with demethylating agents

  • Assess re-expression as a potential biomarker for treatment efficacy

  • Correlate RASSF2 levels with response to targeted therapies

• Multiplex Analysis:

  • Combine FITC-conjugated RASSF2 antibody with antibodies against other pathway components (MST1/2, K-Ras)

  • Assess co-expression patterns in clinical samples

  • Correlate with activation status of downstream pathways (e.g., Hippo, NF-κB)

Validation Studies:

• Include normal tissue controls to establish baseline expression

• Use cell lines with known RASSF2 status as technical controls

• Perform parallel analysis using alternative methods (IHC, Western blot) for confirmation