RASSF4 Antibody

What is RASSF4 and what biological roles does it play?

RASSF4 (Ras Association Domain Family Member 4) belongs to the RASSF family proteins which interact with Ras protein. It functions primarily as a tumor suppressor gene that directly interacts with and activates K-Ras in a GTP-dependent manner . Current research indicates that RASSF4 is involved in multiple biological processes including:

• Inhibition of cell proliferation in cancer cells

• Induction of apoptosis in a Ras-dependent manner

• Increasing sensitivity to chemotherapeutic agents such as 5-Fluorouracil (5-FU) in colorectal cancer

• Regulation of the Hippo signaling pathway through interaction with MST1

• Participation in skeletal muscle differentiation

RASSF4 has been found to be downregulated in multiple cancer types, including colorectal cancer, nasopharyngeal carcinoma, gastric cardia adenocarcinoma, multiple myeloma, and non-small cell lung cancers .

What experimental techniques commonly employ RASSF4 antibodies?

RASSF4 antibodies have been validated for several experimental applications:

Most research laboratories employ a combination of these techniques to validate findings across multiple platforms .

How should RASSF4 antibodies be validated before experimental use?

Proper validation of RASSF4 antibodies is crucial for ensuring reliable results. Based on recommendations from the Working Group for Antibody Validation and practices employed in recent studies, a comprehensive validation approach should include:

• Genetic validation: Using RASSF4 knockdown or knockout models via CRISPRi or siRNA technology to confirm antibody specificity .

• Recombinant expression: Creating stable cell lines overexpressing RASSF4 fused to a fluorescent protein reporter and comparing antibody binding between overexpressing and control cells .

• Orthogonal validation: Correlating protein detection with RASSF4 mRNA levels via RT-qPCR .

• Independent antibody verification: Testing multiple antibodies against different epitopes of RASSF4 to confirm consistent results .

In documented validations, researchers have successfully employed CRISPRi knockdown of RASSF4 in MCF10A KRAS G12V cells to demonstrate antibody specificity, showing significant staining on wild-type cells and minimal binding to the corresponding knockdown cell line .

What are the optimal protocols for RASSF4 detection in immunohistochemistry?

For optimal RASSF4 detection in paraffin-embedded tissue sections, the following protocol has been shown to yield reliable results:

• Deparaffinization and rehydration: Use xylene for deparaffinization followed by graded alcohol for rehydration .

• Antigen retrieval: Perform in 0.01 M Citrate buffer. This step is critical for exposing antigenic epitopes masked during fixation .

• Blocking steps:

  • Treat with hydrogen peroxide to block endogenous peroxidase activity

  • Apply goat serum to reduce non-specific binding

• Primary antibody incubation: Use RASSF4 antibody at 1:300 dilution and incubate overnight at 4°C .

• Detection system: Employ Elivision Super kit (or equivalent) for secondary antibody incubation, followed by DAB plus kit for visualization .

Evaluation of staining should follow the immunoreactive score system, which accounts for both the intensity and percentage of positive cells .

How can RASSF4 antibodies be utilized to investigate its role in the Hippo pathway?

RASSF4 functions in the Hippo pathway by interacting with MST1 to inhibit YAP nuclear translocation. To investigate this interaction:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate FLAG-RASSF4 using M2-agarose following lysis in buffer containing 50mM Tris HCl (pH 7.4), 150mM NaCl, 1mM EDTA, and 1% Triton X-100

  • Western blot analysis of the precipitate using antibodies against MST1 (Cell Signaling, 3682) can confirm interaction

• Immunofluorescence co-localization:

  • RASSF4 and MST1 co-localization can be visualized using antibodies against both proteins

  • In differentiated myotubes, endogenous RASSF4 has been observed to accumulate in a ring-like pattern around nuclei, co-localizing with PCM-1

• Analysis of YAP nuclear translocation:

  • Nuclear and cytoplasmic fractionation followed by Western blot can quantify YAP localization

  • Immunofluorescence staining using antibodies against RASSF4 and YAP can visually demonstrate how RASSF4 affects YAP nuclear translocation

Research has shown that RASSF4 overexpression attenuates YAP nuclear translocation, while RASSF4 knockdown increases it, confirming its role in the Hippo pathway .

What experimental approaches using RASSF4 antibodies can help understand chemotherapy resistance mechanisms?

RASSF4 has been implicated in modulating chemotherapy sensitivity, particularly to 5-FU in colorectal cancer and sorafenib in HCC. Several approaches using RASSF4 antibodies can elucidate these mechanisms:

• Expression correlation studies:

  • Compare RASSF4 expression levels in sensitive versus resistant cancer cell lines using Western blot

  • Correlate RASSF4 expression with patient response to chemotherapy in clinical samples using immunohistochemistry

• Mechanism investigation:

  • Following RASSF4 overexpression or knockdown, analyze changes in mitochondrial membrane potential using JC-1

  • Perform Western blot analysis of apoptosis-related proteins (particularly Bcl-2) and cell cycle regulators (p21)

  • Conduct ChIP assays to evaluate TEAD4 binding to Bcl-2 promoter regions

• Pathway analysis:

  • Use pulldown assays with RASSF4 antibodies followed by mass spectrometry to identify interaction partners in chemoresistant cells

  • Perform Western blot analysis of key Hippo pathway components (MST1/2, YAP/p-YAP) in RASSF4-manipulated cells

Research demonstrates that RASSF4 overexpression increases 5-FU-induced apoptosis and downregulates mitochondrial membrane potential, while RASSF4 knockdown promotes resistance, suggesting its utility as a biomarker for treatment response .

How can researchers optimize co-immunoprecipitation protocols with RASSF4 antibodies?

For successful co-immunoprecipitation of RASSF4 and its interaction partners:

• Lysis buffer optimization:

  • Use a buffer containing 50mM Tris HCl (pH 7.4), 150mM NaCl, 1mM EDTA, and 1% Triton X-100

  • Include protease and phosphatase inhibitors to prevent protein degradation and modification

• Antibody selection:

  • For tagged RASSF4, M2-agarose for FLAG-tagged proteins has shown high efficiency

  • For endogenous RASSF4, select antibodies validated for immunoprecipitation applications

• Binding and elution conditions:

  • Allow binding to occur at 4°C with gentle rotation for 2-4 hours

  • For tagged proteins, elution can be performed using 0.1M glycine HCl (pH 3.5) into tubes containing 1M Tris (pH 8.0) to neutralize the pH

  • For native IP, use protein A/G beads with appropriate species-matched antibodies

• Controls and validation:

  • Always include IgG control precipitations

  • Confirm interactions through reciprocal IP (i.e., precipitate the suspected binding partner and probe for RASSF4)

  • Validate interactions through additional methods such as proximity ligation assay or FRET

Researchers have successfully used these approaches to demonstrate that RASSF4 interacts specifically with MST1 but not MST2, highlighting the importance of proper controls in distinguishing between closely related interaction partners .

What techniques can be used to investigate RASSF4's interaction with the Ras pathway?

To study RASSF4's interaction with Ras proteins:

• Ras activation-dependent binding assays:

  • Use GST-RBD (Ras-binding domain) pulldown assays with RASSF4 antibodies to detect GTP-loaded Ras

  • Compare RASSF4 binding to wild-type versus mutant (G12V) KRAS using co-immunoprecipitation

• Small molecule competition assays:

  • Anti-RASSF4 antibodies can be used as competitors in small-molecule library screens to identify compounds that disrupt Ras-RASSF4 interactions

  • Employ ELISA-based binding assays with immobilized RASSF4 and labeled Ras proteins to measure disruption by small molecules

• Structural studies:

  • Antibody-based protein purification can facilitate crystallography or cryo-EM studies of RASSF4-Ras complexes

  • Epitope mapping using antibody fragments can identify key binding interfaces

• Functional validation:

  • Measure changes in downstream Ras signaling pathways (MAPK, PI3K) in cells with RASSF4 overexpression or knockdown

  • Use proximity ligation assays to visualize endogenous Ras-RASSF4 interactions in intact cells

Research indicates that RASSF4 directly interacts with and activates K-Ras in a GTP-dependent manner, inducing apoptosis in a Ras-dependent fashion .

What are the specific considerations when using RASSF4 antibodies in different tissue types?

RASSF4 expression and detection vary across tissue types, requiring specific optimization:

• Colorectal tissues:

  • Strong RASSF4 staining is typically observed in normal colorectal epithelial tissues

  • In CRC tissues, expression is frequently downregulated (38.2% of cases show low expression)

  • Antigen retrieval using citrate buffer is critical for these tissues

• Liver tissues:

  • RASSF4 is significantly downregulated in MASH and HCC samples

  • Background staining can be problematic; extended blocking steps may be necessary

  • Expression correlates with fibrosis and steatosis markers

• Skeletal muscle:

  • RASSF4 shows transient upregulation during myogenic differentiation

  • Highest expression is observed in early differentiation HSMMs (day three)

  • In differentiated myotubes, RASSF4 localizes in a distinctive ring-like pattern around nuclei

• Cancer cell lines:

  • Expression varies significantly; LoVo cells show lower RASSF4 levels compared to other CRC cell lines

  • HepG2 and MHCC-97H cells show differential expression and can be used as positive/negative controls

When comparing expression across tissues, consistent fixation protocols and antibody concentrations should be maintained to allow for valid quantitative comparisons.

How can RASSF4 antibodies be used to study the relationship between RASSF4 expression and clinical outcomes?

RASSF4 antibodies have proven valuable in investigating correlations between expression and clinical parameters:

Studies have demonstrated that RASSF4 downregulation significantly associates with advanced TNM stage, T status, positive node status, and high Ki-67 index in CRC patients, suggesting its utility as a prognostic biomarker .

What are common challenges in RASSF4 antibody applications and how can they be addressed?

Researchers frequently encounter the following challenges when working with RASSF4 antibodies:

• Non-specific binding:

  • Problem: Background staining in immunohistochemistry or multiple bands in Western blot

  • Solution: Increase blocking time (use 5% BSA or 10% serum), optimize antibody dilution, and include appropriate negative controls (RASSF4 knockdown cells)

• Low signal intensity:

  • Problem: Weak or undetectable RASSF4 signal despite proper technique

  • Solution: Optimize antigen retrieval (try different buffers and heating times), increase antibody concentration, use signal amplification systems, or try alternative antibodies targeting different epitopes

• Inconsistent results across samples:

  • Problem: Variable staining between experiments or tissue sections

  • Solution: Standardize fixation protocols, processing times, and antibody incubation conditions; include positive control tissues in each batch

• Cross-reactivity with other RASSF family members:

  • Problem: Potential detection of homologous proteins (RASSF1-6)

  • Solution: Validate antibody specificity using overexpression and knockdown controls for multiple RASSF family members; consider using more specific monoclonal antibodies

• Detection in fixed versus frozen tissues:

  • Problem: Antibodies may perform differently in frozen versus FFPE tissues

  • Solution: Validate each antibody specifically for the intended application and tissue preparation method

How should researchers approach quantification of RASSF4 immunostaining in research and clinical specimens?

Accurate quantification of RASSF4 immunostaining requires systematic approaches:

• Immunoreactive scoring systems:

  • Use established scoring systems that account for both staining intensity and percentage of positive cells

  • Example system: Multiply intensity score (0-3) by percentage score (0-4) for a final score range of 0-12

  • Scores can be dichotomized as "high" versus "low" expression based on median values or established cutoffs

• Digital image analysis:

  • Use software platforms capable of calculating H-scores or automated quantification

  • Standardize image acquisition parameters (exposure time, white balance)

  • Set consistent thresholds for positive staining across all analyzed samples

• Statistical validation:

  • Perform interobserver and intraobserver validation with multiple trained pathologists

  • Calculate kappa coefficients to ensure scoring consistency

  • Consider using continuous variables for more sensitive statistical analyses

• Controls for normalization:

  • Include internal positive controls in each staining batch

  • Consider normalizing to housekeeping proteins in Western blot or to reference tissues in IHC

  • For comparative studies, process all samples simultaneously when possible

In published studies, researchers have successfully used these approaches to demonstrate that RASSF4 protein expression was downregulated in 38.2% of CRC specimens and that this downregulation correlated significantly with clinical outcomes .