RALA Antibody, FITC conjugated

What is RALA and what are its primary cellular functions?

RALA (v-ral simian leukemia viral oncogene homolog A) is a multifunctional GTPase belonging to the small GTPase superfamily with a molecular weight of approximately 24 kDa. This protein is involved in numerous cellular processes including gene expression, cell migration, cell proliferation, oncogenic transformation, and membrane trafficking . RALA accomplishes these diverse functions through interactions with distinct downstream effectors .

During cell division, RALA plays a critical role in the early stages of cytokinesis by supporting the stabilization and elongation of the intracellular bridge between dividing cells . It cooperates with EXOC2 to recruit other components of the exocyst to the early midbody . Additionally, RALA controls mitochondrial fission during mitosis by recruiting RALBP1 to the mitochondrion, which mediates the phosphorylation and activation of DNM1L by the mitotic kinase cyclin B-CDK1 .

What experimental applications are RALA antibodies typically used for?

RALA antibodies have been validated for multiple experimental applications across different tissue and cell types. Based on comprehensive testing, these antibodies demonstrate utility in:

For optimal results, antibody dilutions should be titrated specifically for each experimental system and sample type .

How does FITC conjugation impact antibody performance in RALA detection?

FITC (Fluorescein isothiocyanate) conjugation provides direct visualization capabilities for RALA antibodies without requiring secondary antibody incubation steps. While the search results don't specifically address FITC-conjugated RALA antibodies, general principles of fluorophore conjugation apply. The FITC molecule (MW ~389 Da) attaches to primary amines on antibodies, potentially affecting binding kinetics depending on conjugation sites relative to the antigen-binding region.

For indirect immunofluorescence applications, FITC-conjugated secondary antibodies have been successfully employed at 1:80 dilutions to detect primary anti-RALA antibodies in fixed HepG2 cells . This demonstrates compatibility of RALA epitopes with FITC-based detection systems.

What are recommended fixation and permeabilization protocols for RALA immunofluorescence studies?

For optimal RALA immunofluorescence detection, a sequential methanol-acetone fixation protocol has been validated:

• Grow cells on appropriate coverslips or slides

• Fix cells for 5 minutes at -20°C using 100% methanol

• Permeabilize for 3 minutes at -20°C using 100% acetone

• Proceed with blocking and antibody incubation

This protocol has been successfully employed with HepG2 cells for RALA detection using both monoclonal antibodies (at 1:2000 dilution) and human sera containing anti-RALA antibodies (at 1:200 dilution) . The methanol-acetone fixation preserves RALA epitopes while providing sufficient permeabilization for antibody access to intracellular targets.

How should samples be prepared for Western blot analysis of RALA?

For Western blot detection of RALA, the following methodology has been validated:

• Separate protein samples using SDS-PAGE

• Transfer proteins to nitrocellulose membrane

• Block with PBS containing 5% non-fat dry milk and 0.05% Tween-20 (PBST) for 30 minutes at room temperature

• Incubate with primary RALA antibody (1:500-1:50000 dilution depending on antibody)

• Apply HRP-conjugated secondary antibody (1:3000 dilution)

• Detect using ECL chemiluminescence reagents

This approach has successfully detected the 24 kDa RALA protein in multiple cell and tissue types, including brain tissues from human, mouse, and rat sources, as well as MCF-7, HeLa, HepG2, and Jurkat cell lines .

What controls should be included when performing RALA activation assays?

When assessing RALA activation status using methods such as the RalA G-LISA Activation Assay, several controls are essential:

• Negative control: Include lysates from serum-starved cells to establish baseline activation

• Positive control: Use lysates from cells stimulated with known RALA activators (e.g., EGF at 100 ng/ml for 2 minutes)

• Loading control: Normalize protein concentration (recommended 12.5-25 μg/well)

• GTP/GDP loading controls: Include extracts artificially loaded with either GDP (inactive) or GTP (active) to determine the maximal activation window

• Blank control: Include buffer-only wells to establish background signal

Experimental data shows that this approach provides a clear activation window, with absorbance readings at 490 nm demonstrating significant differences between serum-starved and EGF-stimulated Rat-2 cells .

How can non-specific binding be minimized when using RALA antibodies?

To reduce non-specific binding when working with RALA antibodies, implement these evidence-based strategies:

• Optimize blocking: Use 5% non-fat dry milk in PBS with 0.05% Tween-20 for Western blotting applications

• Antibody titration: Determine optimal concentration through serial dilutions; recommended starting ranges:

  • WB: 1:500-1:50000

  • IHC: 1:50-1:2000

  • FC: 0.40 μg per 10^6 cells

• Buffer optimization: For antigen retrieval in IHC, test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) to determine which provides optimal signal-to-noise ratio

• Pre-absorption controls: When validating specificity, pre-absorb antibodies with the target antigen to confirm signal elimination

These approaches have been validated across multiple RALA antibody applications and sample types, ensuring reliable and specific detection.

What factors influence RALA epitope accessibility in fixed tissues?

Several factors affect RALA epitope accessibility in fixed tissue samples:

• Fixation method: RALA epitopes are sensitive to fixation conditions. For IHC applications, formalin-fixed paraffin-embedded tissues require specific antigen retrieval approaches.

• Antigen retrieval: Two buffer systems have proven effective:

  • TE buffer (pH 9.0) - recommended as primary choice

  • Citrate buffer (pH 6.0) - alternative approach

• Tissue type: Different tissues may require adjusted protocols. RALA antibodies have been successfully used with:

  • Human breast cancer tissue

  • Liver tissues (normal, cirrhotic, and HCC)

  • Brain tissues from multiple species

• Subcellular localization: RALA shuttles between cellular compartments depending on activation state, potentially affecting epitope accessibility

When optimizing protocols, these variables should be systematically evaluated to ensure consistent and specific RALA detection.

How can RALA antibodies be used to assess activation states in signaling pathways?

RALA cycles between inactive GDP-bound and active GTP-bound states, with only the GTP-bound form interacting with downstream effectors. The RalA G-LISA Activation Assay provides a specialized approach for quantifying active RALA:

• The assay uses a plate coated with Ral-GTP-binding protein that selectively captures active GTP-bound RALA

• Inactive GDP-bound RALA is removed through washing steps

• Bound active RALA is detected using RALA-specific antibodies

• Colorimetric detection provides quantitative measurement with absorbance readings at 490 nm

• The assay is linear from 0.5 to 5 ng of protein and can detect activation differences between serum-starved and growth factor-stimulated cells

This methodology has successfully demonstrated RALA activation in response to EGF stimulation in Rat-2 cells, showing approximately 2-fold activation following treatment with 100 ng/ml EGF for 2 minutes .

What is the relationship between RALA expression and hepatocellular carcinoma progression?

RALA demonstrates a significant relationship with hepatocellular carcinoma (HCC) development and progression. Immunohistochemical studies using tissue microarrays have revealed a stepwise increase in RALA expression across the progression from normal liver to HCC:

Additionally, autoantibody responses against RALA show a distinctive pattern:

These findings suggest RALA may contribute to liver malignant transformation and could potentially serve as a tumor marker in HCC detection with a sensitivity of 20.1% and a specificity of 99.3% .

How can multiplexed immunofluorescence approaches incorporate RALA detection?

While the search results don't specifically address multiplexed immunofluorescence with RALA antibodies, methodological principles can be derived from the documented immunofluorescence protocols. For developing multiplexed approaches:

• Fluorophore selection: FITC (excitation ~495 nm, emission ~519 nm) can be combined with fluorophores having minimal spectral overlap, such as:

  • TRITC/Cy3 (excitation ~550 nm, emission ~570 nm)

  • Cy5 (excitation ~650 nm, emission ~670 nm)

• Sequential staining: To avoid antibody cross-reactivity:

  • Apply the first primary antibody (e.g., RALA)

  • Detect with fluorophore-conjugated secondary antibody

  • Block remaining binding sites

  • Apply subsequent primary and secondary antibodies

• Fixation compatibility: The validated methanol-acetone fixation protocol for RALA detection should be assessed for compatibility with other target proteins in multiplexed approaches

• Advanced imaging: Confocal laser-scanning microscopy has been successfully employed for RALA detection in HepG2 cells and provides the resolution needed for colocalization studies in multiplexed applications

What role does RALA play in cancer cell proliferation and transformation?

RALA contributes to cancer development through multiple mechanisms:

• Anchorage-independent growth: RALA is required for anchorage-independent proliferation of transformed cells, a hallmark of cancer

• Signaling pathway regulation: RALA functions as a key regulator of LPAR1 signaling and competes with GRK2 for binding to LPAR1, affecting receptor signaling properties

• Exocyst complex regulation: The RALA-exocyst complex regulates integrin-dependent membrane raft exocytosis and growth signaling

• Progressive upregulation: The stepwise increase in RALA expression from normal liver (26.7%) to liver cirrhosis (45.0%) to HCC (63.3%) suggests its involvement in malignant transformation

These functions position RALA as a potential therapeutic target and diagnostic marker in cancer research, particularly in hepatocellular carcinoma where it demonstrates both increased expression and immunogenicity .

How should experiments be designed to evaluate RALA as a potential biomarker?

Based on existing research demonstrating RALA's potential as a biomarker in HCC, experimental designs should include:

• Multi-modal detection approaches:

  • Tissue expression analysis via IHC

  • Serum autoantibody detection via ELISA

  • Protein activation assays via G-LISA

• Comprehensive control groups:

  • Disease progression controls (e.g., normal tissue, pre-malignant conditions, cancer)

  • Disease specificity controls (various cancer types)

  • Demographic-matched healthy controls

• Sensitivity and specificity determination:

  • Establish cutoff values based on receiver operating characteristic (ROC) analysis

  • Calculate sensitivity, specificity, positive predictive value, and negative predictive value

  • In HCC research, anti-RALA antibody detection demonstrated 20.1% sensitivity and 99.3% specificity

• Correlation with clinical parameters:

  • Disease stage

  • Treatment response

  • Survival outcomes

This comprehensive approach aligns with the methodologies that established RALA's potential biomarker utility in hepatocellular carcinoma research.