RNF19A Antibody, FITC conjugated

What is RNF19A and what cellular functions does it perform?

RNF19A (Ring Finger Protein 19A) is a member of the RING-in-between-RING (RBR) E3 ubiquitin ligase family. It functions primarily in the ubiquitin-proteasome pathway, targeting proteins for degradation through ubiquitination. Recent research has revealed that RNF19A plays critical roles in cancer progression through various mechanisms. In non-small cell lung cancer (NSCLC), RNF19A mediates p53 ubiquitin-degradation, promoting cancer growth by reducing p53, p21, and BAX expression while inducing Cyclin D1, CDK4, CDK6, and BCL2 expression . Conversely, in bladder cancer, RNF19A acts as a tumor suppressor by regulating ILK and inactivating the AKT/mTOR signaling pathway .

The protein contains specialized domains that facilitate its E3 ligase function, including RING finger domains that are essential for the ubiquitination process. These structural characteristics make it an important target for both basic research and potential therapeutic development.

How does RNF19A expression vary across different tissues and cell types?

RNF19A demonstrates broad cross-reactivity across multiple species. According to antibody validation studies, RNF19A shows 100% predicted reactivity in humans, mice, cows, dogs, and horses, with slightly lower reactivity (93%) in guinea pigs and rabbits . This conservation across species suggests fundamental biological roles.

What epitopes are targeted by common RNF19A antibodies and how does this affect experimental applications?

RNF19A antibodies target different regions of the protein depending on the specific product. The search results reveal several antibody options:

• N-terminal targeting antibodies: These recognize epitopes at the amino-terminal region of RNF19A, such as the synthetic peptide "IFSTNTSSDN GLTSISKQIG DFIECPLCLL RHSKDRFPDI MTCHHRSCVD"

• Mid-region targeting antibodies: Some antibodies target amino acids 108-157

• C-terminal targeting antibodies: Others recognize the carboxy-terminal region (AA 741-825)

Epitope selection significantly impacts experimental outcomes. N-terminal antibodies may detect full-length RNF19A but might miss truncated variants. C-terminal antibodies can identify both full-length and C-terminal fragments but might miss N-terminal fragments. For comprehensive detection of all RNF19A forms, researchers should consider using multiple antibodies targeting different regions.

What are the key considerations when selecting FITC-conjugated RNF19A antibodies?

When selecting FITC-conjugated RNF19A antibodies, researchers should consider:

• Epitope specificity: Choose antibodies recognizing relevant epitopes based on research questions (N-terminal vs C-terminal)

• Validation documentation: Look for antibodies validated in applications similar to your planned experiments

• Species reactivity: Ensure compatibility with your experimental model (human, mouse, etc.)

• Clonality: Polyclonal antibodies provide broader epitope recognition, while monoclonal antibodies offer higher specificity

• Fluorophore characteristics: FITC excites at ~495nm and emits at ~519nm, making it compatible with standard FITC filter sets but susceptible to photobleaching

For FITC-conjugated RNF19A antibodies specifically, consider using those targeting amino acids 741-825, as these have been documented in the search results to be available with FITC conjugation . This region appears to be important for RNF19A function and provides good detection sensitivity.

What are the optimal protocols for immunofluorescence using FITC-conjugated RNF19A antibodies?

Optimized Immunofluorescence Protocol for FITC-conjugated RNF19A Antibodies:

• Sample preparation:

  • For cell cultures: Grow cells on coverslips to 60-70% confluence

  • For tissue sections: Use freshly frozen or formalin-fixed paraffin-embedded sections (5-7μm thickness)

• Fixation:

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

  • For tissues: After deparaffinization and rehydration, perform antigen retrieval (citrate buffer pH 6.0, 95°C for 20 minutes)

• Permeabilization and blocking:

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

  • Block with 5% normal serum (matching secondary antibody species) with 1% BSA in PBS for 1 hour

• Primary antibody incubation:

  • Dilute FITC-conjugated RNF19A antibody at 1:200-1:500 as recommended

  • Incubate overnight at 4°C in a humidified chamber protected from light

• Nuclear counterstaining:

  • Counterstain with DAPI (1μg/ml) for 5 minutes

  • Mount with anti-fade mounting medium

• Controls:

  • Include a negative control (omitting primary antibody)

  • For specificity validation, include samples with known RNF19A expression levels

Researchers should optimize this protocol for their specific experimental conditions, including adjusting antibody concentration, incubation time, and washing steps based on signal intensity and background levels.

How can RNF19A-FITC antibodies be effectively used in studying protein interactions?

FITC-conjugated RNF19A antibodies are valuable tools for studying protein-protein interactions through techniques such as:

Proximity Ligation Assays (PLA):

• Culture cells or prepare tissue sections as appropriate

• Fix and permeabilize samples

• Block with appropriate blocking solution

• Incubate with FITC-conjugated RNF19A antibody and an antibody against a potential interacting protein

• Follow PLA protocol with appropriate PLA probes

• Analyze the proximity signals indicating protein interaction

Co-immunoprecipitation with RNF19A:
Based on the research showing RNF19A interactions with proteins like p53 and ILK , the following protocol can be adapted:

• Prepare cell lysates using RIPA buffer containing protease inhibitors

• Pre-clear lysates with protein A/G beads

• Immunoprecipitate with anti-RNF19A antibody overnight at 4°C

• Add protein A/G beads and incubate for 2-4 hours

• Wash beads thoroughly and elute proteins

• Analyze by Western blot using antibodies against suspected interaction partners

In bladder cancer research, RNF19A was found to directly interact with ILK and promote its ubiquitination and degradation . Similar approaches can be used to investigate other potential RNF19A interacting partners.

How does RNF19A expression influence cancer progression and clinical outcomes?

RNF19A appears to have context-dependent roles in cancer progression, with opposing functions in different cancer types:

Non-Small Cell Lung Cancer (NSCLC):

• RNF19A is significantly overexpressed in NSCLC tissues compared to normal lung tissues

• Higher RNF19A expression correlates with poor clinical outcomes in NSCLC patients

• RNF19A promotes cancer cell proliferation and inhibits apoptosis in NSCLC

• Mechanistically, RNF19A reduces expression of tumor suppressors (p53, p21, BAX) and increases expression of oncogenes (Cyclin D1, CDK4, CDK6, BCL2)

Bladder Cancer (BCa):

These contrasting roles highlight the importance of tissue-specific context in understanding RNF19A function and suggest that therapeutic strategies targeting RNF19A would need to be cancer type-specific.

What is the mechanism of RNF19A-mediated p53 regulation in cancer?

RNF19A regulates p53 through direct protein-protein interaction and subsequent ubiquitin-mediated degradation. The detailed mechanism, as revealed in NSCLC research, includes:

• Physical interaction: RNF19A directly binds to p53 protein, as demonstrated through co-immunoprecipitation experiments

• Reduced p53 half-life: RNF19A shortens the half-life of p53 protein

• Ubiquitination: As an E3 ubiquitin ligase, RNF19A mediates p53 ubiquitination, marking it for proteasomal degradation

• Downstream effects: Reduced p53 levels lead to decreased expression of p53 target genes (p21, BAX) and increased expression of cell cycle regulators (Cyclin D1, CDK4, CDK6) and anti-apoptotic factors (BCL2)

This mechanism explains how RNF19A overexpression promotes cancer progression in NSCLC through inhibition of p53-mediated tumor suppression. The functional significance of this regulation was confirmed by rescue experiments showing that p53 silencing partially reversed the inhibitory effects of RNF19A knockdown on cancer cell proliferation .

What are effective approaches for establishing RNF19A knockdown or overexpression models?

Lentiviral-Mediated RNF19A Knockdown Protocol:

• Design and construction:

  • Design shRNA sequences targeting RNF19A (e.g., targeting regions with high conservation)

  • Clone into appropriate lentiviral vectors (e.g., pLKO.1)

  • Include appropriate controls (scrambled shRNA)

• Lentivirus production:

  • Transfect packaging cells (HEK293T) with lentiviral vector plus packaging plasmids

  • Collect virus-containing supernatant 48-72h post-transfection

  • Filter through 0.45μm filter

• Target cell infection:

  • Plate target cells at 40-50% confluence in 6-well plates

  • Add appropriate amount of lentivirus with polybrene (8μg/ml)

  • Incubate for 12 hours, then replace medium with fresh complete medium containing 20% PBS

  • Select stable cells with puromycin (3μg/ml) after 36 hours

• Validation:

  • Confirm knockdown efficiency by qRT-PCR and Western blot

  • For qRT-PCR of RNF19A, use primers: Forward 5'-CCATCCGAGACAACCTGAGT-3', Reverse 5'-ACTGTTCCCAAGCTGACTGT-3'

Lentiviral-Mediated RNF19A Overexpression Protocol:
Similar to the knockdown approach, but using a lentiviral vector containing the RNF19A coding sequence. Consider using a FLAG-tagged RNF19A construct to facilitate detection and immunoprecipitation experiments .

What methods are most reliable for studying RNF19A-mediated protein ubiquitination?

In-Cell Ubiquitination Assay Protocol:

• Plasmid transfection:

  • Transfect cells with:

    • Flag-tagged RNF19A expression plasmid

    • HA-tagged target protein (e.g., ILK or p53)

    • His-tagged ubiquitin expression plasmid

  • Use appropriate transfection reagent (e.g., HighGene reagent)

  • Include appropriate controls (empty vector controls)

• Treatment:

  • 44-48 hours post-transfection, treat cells with proteasome inhibitor (MG132, 10μM) for 4-6 hours

• Cell lysis:

  • Lyse cells in denaturing buffer (6M guanidine-HCl, 0.1M Na2HPO4/NaH2PO4, 10mM imidazole, pH 8.0)

• Pulldown of ubiquitinated proteins:

  • Incubate lysates with Ni-NTA beads for 3-4 hours at room temperature

  • Wash beads extensively with washing buffers of decreasing stringency

  • Elute ubiquitinated proteins with elution buffer containing imidazole

• Analysis:

  • Separate proteins by SDS-PAGE

  • Perform Western blot analysis using antibodies against the tag on your target protein

This protocol allows detection of ubiquitinated target proteins while ensuring specificity through the use of tagged proteins and denaturing conditions that disrupt non-covalent interactions.

What are the most common issues with FITC-conjugated antibodies and how can they be resolved?

Common Issue 1: Photobleaching

• Problem: FITC is susceptible to photobleaching during extended imaging sessions

• Solution: Add anti-fade reagents to mounting medium, minimize exposure to excitation light, use lower intensity for excitation, and consider taking images of control samples first to standardize exposure settings

Common Issue 2: High Background Fluorescence

• Problem: Non-specific binding or autofluorescence masking specific signals

• Solution:

  • Optimize blocking (try different blocking agents: 5% BSA, normal serum, or commercial blocking reagents)

  • Include 0.1-0.3% Triton X-100 in antibody diluent

  • Increase washing steps (at least 3x5 minutes with PBS-T)

  • Use Sudan Black B (0.1% in 70% ethanol) to reduce autofluorescence

  • Consider shorter primary antibody incubation time or more dilute antibody solution

Common Issue 3: Weak or No Signal

• Problem: Insufficient antibody binding or epitope accessibility

• Solution:

  • Optimize antibody concentration (test range from 1:100 to 1:500)

  • Ensure proper antigen retrieval for fixed tissues

  • Extend primary antibody incubation time (overnight at 4°C)

  • Check that storage conditions haven't compromised antibody activity

  • Verify that target protein is expressed in your samples

What controls should be included to validate RNF19A antibody specificity?

Essential Controls for RNF19A Antibody Validation:

• Negative Controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype control: Use non-specific IgG from the same species as primary antibody

  • Blocking peptide competition: Pre-incubate antibody with immunizing peptide before application

• Positive Controls:

  • Cell lines or tissues with known RNF19A expression

  • Overexpression system: Cells transfected with RNF19A expression vector

  • For NSCLC studies, lung cancer cell lines with confirmed RNF19A expression

  • For bladder cancer studies, appropriate bladder cancer cell lines

• Knockdown/Knockout Controls:

  • RNF19A knockdown cells created using shRNA or siRNA

  • CRISPR/Cas9-mediated RNF19A knockout cells

  • These controls should show reduced or absent staining

• Multiple Antibody Validation:

  • Use multiple antibodies targeting different epitopes of RNF19A

  • Compare staining patterns between antibodies targeting N-terminal (ABIN2774733) and C-terminal regions (ABIN7151172)

How should researchers quantify and interpret RNF19A expression data in cancer studies?

Quantification Methods for RNF19A Expression:

• Western Blot Quantification:

  • Normalize RNF19A band intensity to loading controls (GAPDH, β-actin)

  • Use software like ImageJ for densitometry analysis

  • Present data as relative expression (fold change) compared to control samples

• Immunofluorescence Quantification:

  • Capture images using standardized exposure settings

  • Measure mean fluorescence intensity within regions of interest

  • Quantify multiple fields (>5) and cells (>100) per sample

  • Subtract background fluorescence from non-specific regions

  • Present data as mean fluorescence intensity or integrated density

• qRT-PCR Analysis:

  • Use validated primers for RNF19A: Forward 5'-CCATCCGAGACAACCTGAGT-3', Reverse 5'-ACTGTTCCCAAGCTGACTGT-3'

  • Normalize to stable reference genes (GAPDH, ACTB)

  • Calculate relative expression using the comparative Ct method (ΔΔCT)

Interpretation Guidelines:

What are emerging areas of RNF19A research that could benefit from FITC-conjugated antibodies?

• Single-Cell Analysis of RNF19A Expression:

  • FITC-conjugated RNF19A antibodies could enable flow cytometry and cell sorting based on RNF19A expression

  • This would facilitate investigation of RNF19A expression heterogeneity within tumors

  • Potential to identify and characterize RNF19A-high and RNF19A-low subpopulations within cancer tissues

• Live Cell Imaging of RNF19A Dynamics:

  • Using cell-permeable FITC-conjugated antibody fragments to monitor RNF19A localization in real-time

  • Studying RNF19A redistribution in response to stress, treatment, or cell cycle progression

  • Correlating dynamic changes with functional outcomes

• Multiplex Imaging Approaches:

  • Combining FITC-RNF19A antibodies with antibodies against interaction partners (p53, ILK)

  • Using multiplexed imaging to study co-localization and pathway activation states

  • Correlating RNF19A with markers of ubiquitination and proteasomal degradation

• Therapeutic Target Validation:

  • Leveraging FITC-conjugated antibodies to monitor RNF19A expression changes in response to potential inhibitors

  • Screening compounds that modulate RNF19A expression or activity

  • Development of antibody-drug conjugates targeting RNF19A-expressing cells

How might advanced microscopy techniques enhance RNF19A research using FITC-conjugated antibodies?

Super-Resolution Microscopy Applications:

• Structured Illumination Microscopy (SIM) to visualize RNF19A co-localization with interaction partners at sub-diffraction resolution

• Stochastic Optical Reconstruction Microscopy (STORM) to map precise RNF19A distribution patterns at nanoscale resolution

• Stimulated Emission Depletion (STED) microscopy to visualize RNF19A in specific subcellular compartments

Correlative Light and Electron Microscopy (CLEM):
Using FITC-conjugated RNF19A antibodies to identify regions of interest for subsequent electron microscopy analysis, providing ultrastructural context for RNF19A localization

Fluorescence Lifetime Imaging Microscopy (FLIM): Exploiting the fluorescence lifetime properties of FITC to detect subtle changes in the microenvironment of RNF19A, potentially indicating conformational changes or protein-protein interactions