rnf144aa Antibody

What is RNF144AA and what is its primary function in cellular processes?

RNF144AA is a 292 amino acid single-pass membrane protein that functions as an E3 ubiquitin-protein ligase. It contains one RING-type zinc finger and two IBR-type zinc fingers, which are critical for its enzymatic activity. The protein accepts ubiquitin from E2 ubiquitin-conjugating enzymes, such as UBC8 and UBCH7, and transfers these ubiquitin residues to target substrates . This ubiquitination process marks proteins for degradation or alters their function, localization, or interactions. Through its RING finger domain, RNF144AA plays important roles in protein-DNA and protein-protein interactions throughout the cell . Studying RNF144AA antibodies helps researchers investigate these ubiquitination pathways and their impact on cellular regulation.

What are the key structural domains of RNF144AA that antibodies typically target?

RNF144AA contains several key structural domains that are often targeted by antibodies:

• RING finger domain: Critical for E3 ligase activity

• IBR-type zinc fingers (two): Important for protein structure and function

• Transmembrane (TM) domain: Highly conserved among species and essential for membrane localization and self-association

• GXXXG motif within the TM domain: Mediates protein self-association

When selecting an RNF144AA antibody, researchers should consider which domain they wish to target based on their experimental goals. For instance, antibodies against the RING finger domain may be useful for studying the protein's ligase activity, while antibodies against the TM domain might help investigate its membrane localization properties.

What are the optimal conditions for using RNF144AA antibodies in Western blot analyses?

For optimal Western blot results with RNF144AA antibodies:

• Sample preparation:

  • Extract proteins using a membrane protein extraction buffer containing mild detergents (e.g., 1% NP-40 or Triton X-100)

  • Include protease inhibitors to prevent degradation

  • For subcellular fractionation studies, separate membrane fractions from cytosolic components

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Load 20-50 μg of total protein per lane

• Transfer conditions:

  • Semi-dry or wet transfer at 100V for 60-90 minutes

  • Use PVDF membranes for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary RNF144AA antibody at 1:500-1:2000 (optimize based on specific antibody)

  • Incubate overnight at 4°C

  • Use appropriate species-specific HRP-conjugated secondary antibody (1:5000-1:10000)

• Detection considerations:

  • RNF144AA typically appears at ~33 kDa, but may show multiple bands due to post-translational modifications

  • Membrane-bound forms may appear in higher molecular weight fractions

When analyzing RNF144AA in different cellular fractions, use markers for membrane (e.g., Na⁺/K⁺-ATPase) and cytosolic (e.g., GAPDH) fractions as controls to validate fractionation.

How can I optimize immunoprecipitation experiments using RNF144AA antibodies?

To optimize immunoprecipitation (IP) of RNF144AA:

• Lysis buffer selection:

  • Use buffers containing 1% NP-40 or 0.5% Triton X-100 with 150 mM NaCl and 50 mM Tris-HCl (pH 7.4)

  • Include protease inhibitors and 1-2 mM EDTA

  • For studying ubiquitination, add deubiquitinase inhibitors (e.g., N-ethylmaleimide)

• Pre-clearing step:

  • Pre-clear lysates with Protein A/G beads (30-60 minutes) to reduce non-specific binding

• Antibody binding:

  • Use 2-5 μg of RNF144AA antibody per 500-1000 μg of protein lysate

  • For co-IP experiments investigating self-association, use tag-specific antibodies (e.g., anti-FLAG) with tagged RNF144AA constructs

  • Incubate with antibody overnight at 4°C with gentle rotation

• Validation controls:

  • Include IgG control from the same species as the primary antibody

  • For studies of self-association, use RNF144AA-ΔTM mutants as negative controls

• Elution and analysis:

  • Elute with 2X SDS sample buffer at 95°C for 5 minutes

  • Analyze by Western blot using a different RNF144AA antibody recognizing a different epitope

For studying RNF144AA self-association, consider using cross-linking agents like dimethyl pimelimidate-2HCl before cell lysis, which has been shown to preserve RNF144A oligomeric structures .

How do RNF144AA expression patterns correlate with other genes in zebrafish models?

RNF144AA expression in zebrafish shows specific correlation patterns with other genes, which can inform functional studies. Analysis of gene expression data reveals:

Positive correlations with:

• Cell adhesion molecules: cldn1 (r=0.066), cldni (r=0.065)

• Extracellular matrix components: col14a1b (r=0.060), col17a1a (r=0.053), col7a1 (r=0.053)

• Signaling molecules: epgn (r=0.056), stmn2a (r=0.053)

• Membrane transport proteins: aqp3a (r=0.056)

• Cytoskeletal components: cotl1 (r=0.060)

Negative correlations with:

• Cell cycle regulators: mki67 (r=-0.040), ccna2 (r=-0.037), ccnb1 (r=-0.036), ccnd1 (r=-0.033)

• DNA replication/repair proteins: lig1 (r=-0.039), pcna (r=-0.035)

• Transcription factors: sox19a (r=-0.035), sox3 (r=-0.035), pou5f3 (r=-0.034)

These correlation patterns suggest potential roles for RNF144AA in:

• Cell adhesion and extracellular matrix organization

• Negative regulation of cell proliferation

• Membrane and cytoskeletal dynamics

When designing zebrafish experiments targeting RNF144AA with antibodies, researchers should consider monitoring these correlated genes to gain deeper insights into pathway interactions. Additionally, immunostaining with RNF144AA antibodies may reveal tissue-specific expression patterns that align with these gene correlation networks.

What is known about the role of the transmembrane domain in regulating RNF144AA function?

The transmembrane (TM) domain of RNF144AA plays crucial roles in regulating its function through two independent mechanisms:

These findings highlight the importance of using domain-specific antibodies when studying RNF144AA function. When designing experiments with RNF144AA antibodies, researchers should consider whether the antibody epitope includes or affects the TM domain, as this could influence experimental outcomes, particularly in studies of protein localization, interaction partners, or E3 ligase activity.

Why might I observe different subcellular localization patterns when using different RNF144AA antibodies?

Discrepancies in RNF144AA localization patterns between different antibodies may result from several factors:

• Epitope accessibility in different cellular compartments:

  • Antibodies targeting the transmembrane domain may show reduced signal in membrane-embedded RNF144AA

  • Conformational changes in different cellular compartments may mask or expose epitopes

• Recognition of specific protein forms:

  • Some antibodies may preferentially detect self-associated versus monomeric forms

  • Antibodies may differentially recognize post-translationally modified forms of RNF144AA

• Fixation-dependent epitope masking:

  • Paraformaldehyde fixation can mask epitopes near the membrane

  • Methanol fixation may better preserve some epitopes but disrupt membrane structures

• Protocol optimization recommendations:

  • Test multiple fixation methods (4% PFA, methanol, or acetone)

  • Include detergent permeabilization optimization (0.1-0.5% Triton X-100 or 0.1% saponin)

  • Use cellular fractionation followed by Western blotting as a complementary approach

  • Consider using GFP-tagged RNF144AA constructs as positive controls

When RNF144AA is properly localized, wild-type protein should appear predominantly in the membrane fraction, while mutants like RNF144A-ΔTM or RNF144A-3L259R should appear in the soluble fraction . Using these constructs as controls can help validate antibody specificity for different subcellular pools of the protein.

How can I distinguish between RNF144AA and RNF144AB proteins in zebrafish models?

Distinguishing between the closely related RNF144AA and RNF144AB proteins in zebrafish requires careful experimental design:

• Sequence comparison and antibody selection:

  • Perform sequence alignment to identify regions of divergence

  • Select antibodies targeting non-conserved regions or validate antibody specificity using knockout/knockdown controls

  • Consider using custom antibodies against unique peptide sequences

• Expression pattern analysis:

  • RNF144AA and RNF144AB may show different temporal or spatial expression patterns

  • Use in situ hybridization with specific probes to map expression domains before antibody studies

• Validation methods:

  • Perform Western blots on tissues from morpholino knockdowns of each gene

  • Use CRISPR/Cas9-generated knockout or knockin reporter lines for each gene

  • Test antibody specificity on overexpressed tagged proteins in cell lines

• Experimental controls to include:

  • Preincubation of antibodies with immunizing peptides to verify specificity

  • Side-by-side comparison with GFP-tagged overexpression constructs

  • Parallel staining with commercial antibodies for both rnf144aa and rnf144ab

• Differential detection strategies:

  • Two-color immunofluorescence with antibodies raised in different species

  • Sequential immunoprecipitation to deplete one protein before detecting the other

  • Mass spectrometry following immunoprecipitation to identify unique peptides

When designing primers or selecting antibodies, focus on regions where sequence homology is lowest between the two proteins to maximize specificity.

What parallels exist between RNF144A and RNF144B in immune regulation?

Recent research has revealed interesting parallels and distinctions between RNF144A and RNF144B in immune regulation:

• Structural similarities:

  • Both belong to the RBR (RING1-IBR-RING2) E3 ubiquitin ligase family

  • Both contain transmembrane domains with GXXXG motifs

  • Both exhibit self-association properties mediated by their TM domains

• Functional distinctions in immune regulation:

  • RNF144B specifically targets MDA5, a crucial cytoplasmic dsRNA sensor

  • RNF144B promotes K27/K33-linked polyubiquitination of MDA5 at lysine 23 and lysine 43

  • This ubiquitination triggers autophagic degradation of MDA5 via p62 recognition

  • RNF144B deficiency enhances type I interferon production and viral resistance

  • RNF144B expression is upregulated during RNA virus infection

• Experimental approaches to study their roles:

  • Use specific antibodies to monitor expression changes during viral infection

  • Employ co-immunoprecipitation to identify interaction partners

  • Utilize in vitro ubiquitination assays to determine substrate specificity

  • Apply CRISPR/Cas9-mediated gene editing to create knockout models

• Potential research applications:

  • Comparing tissue-specific expression of both proteins during immune challenges

  • Investigating whether RNF144A also targets immune signaling components

  • Exploring their relative contributions in different viral infection models

  • Examining potential compensatory mechanisms in single knockout models

While RNF144B has been established as a negative regulator of antiviral immunity , the potential role of RNF144A in immune regulation remains less characterized and represents an exciting frontier for researchers using RNF144A antibodies.

How might RNF144AA antibodies contribute to understanding protein-protein interactions in disease models?

RNF144AA antibodies can be powerful tools for investigating protein-protein interactions in disease contexts:

• Mapping interaction networks in cancer models:

  • The G252D mutation in RNF144A found in human cancers affects protein stability

  • RNF144AA antibodies can help identify altered interaction patterns in mutant versus wild-type contexts

  • Proximity ligation assays using RNF144AA antibodies can visualize interaction changes in situ

• Investigating ubiquitination targets in neurodegenerative diseases:

  • E3 ubiquitin ligases like RNF144AA may influence protein aggregation and clearance

  • Co-immunoprecipitation with RNF144AA antibodies followed by mass spectrometry can identify novel substrates

  • Ubiquitination assays using immunopurified RNF144AA can validate potential targets

• Methodological approaches:

  • BioID or TurboID proximity labeling with RNF144AA as bait

  • FRET/BRET assays using antibody-based detection systems

  • Crosslinking mass spectrometry (XL-MS) to capture transient interactions

  • Super-resolution microscopy with RNF144AA antibodies to visualize nanoscale interaction domains

• Disease model applications:

  • Compare RNF144AA interaction partners between normal and disease states

  • Monitor changes in RNF144AA localization during disease progression

  • Assess RNF144AA expression and interactome changes in response to therapeutic interventions

• Technical considerations:

  • Use multiple antibodies targeting different RNF144AA epitopes to validate interactions

  • Include appropriate controls (IgG, blocking peptides, knockdown/knockout samples)

  • Consider membrane protein-specific interaction methods for membrane-bound RNF144AA

By employing RNF144AA antibodies in these approaches, researchers can gain deeper insights into how this E3 ligase contributes to normal cellular function and disease pathology.

How do antibody responses to RNF144AA compare with other E3 ubiquitin ligases in immunological studies?

When comparing antibody responses to RNF144AA with other E3 ubiquitin ligases:

• Epitope accessibility differences:

  • Membrane-associated E3 ligases like RNF144AA present unique challenges due to transmembrane domains

  • Cytosolic E3 ligases often show more consistent antibody epitope accessibility

  • Comparative immunoprecipitation efficiency:

  E3 Ligase Type	Average IP Efficiency	Key Optimization Factors
  Membrane-associated (RNF144A)	40-60%	Detergent selection, membrane solubilization
  Cytosolic (TRIM family)	70-85%	Salt concentration, pH optimization
  Nuclear (RNF8/RNF168)	50-65%	Nuclear extraction method, chromatin state

• Antibody cross-reactivity considerations:

  • RBR family E3 ligases (including RNF144A/B) show conserved RING domains

  • Antibodies targeting the RING domain may show cross-reactivity

  • Validation techniques for specificity include:

    • Western blotting with multiple family members expressed in parallel

    • Immunoprecipitation followed by mass spectrometry to identify bound proteins

    • Testing in knockout/knockdown models of specific family members

• Structural features affecting antibody binding:

  • RNF144AA self-association may create conformational epitopes absent in monomers

  • Post-translational modifications may mask antibody binding sites

  • Membrane insertion can restrict access to certain domains

• Methodological recommendations:

  • Use antibodies targeting unique regions outside conserved domains when possible

  • Include positive and negative controls from related E3 ligase family members

  • Validate antibody specificity using overexpression and knockdown approaches

  • Consider native versus denaturing conditions when comparing antibody performance

Understanding these comparative aspects helps researchers select and validate appropriate antibodies for studying RNF144AA in relation to other E3 ubiquitin ligases.

What insights from antibody development for RING finger proteins can be applied to RNF144AA research?

Lessons from antibody development for RING finger proteins provide valuable insights for RNF144AA research:

• Epitope selection strategies:

  • Target unique regions outside the conserved RING domain to improve specificity

  • Consider the accessibility of epitopes in the native protein conformation

  • For RNF144AA, regions between the RING and IBR domains or C-terminal to the TM domain often yield more specific antibodies

• Validation approaches from successful RING finger protein antibodies:

  • Use knockout cell lines or tissues as gold standard negative controls

  • Employ peptide competition assays to confirm epitope specificity

  • Test antibodies on denatured and native proteins to assess conformation sensitivity

• Application-specific considerations:

  • For ubiquitination studies: Antibodies against the catalytic RING domain may interfere with enzymatic activity

  • For localization studies: Antibodies against the TM domain may have limited accessibility

  • For interaction studies: Epitopes at protein-protein interfaces may be masked in complexes

• Advanced technological approaches:

  • Single-domain antibodies (nanobodies) have shown success with RING finger proteins due to their small size and ability to access restricted epitopes

  • Proximity-dependent labeling with TurboID or APEX2 fusions can complement antibody-based approaches

  • Conditional protein degradation systems (e.g., AID or dTAG) with epitope tags can overcome antibody limitations

• Experimental design recommendations:

  • Use multiple antibodies targeting different regions of RNF144AA

  • Include domain deletion mutants as controls for epitope mapping

  • Consider generating site-specific antibodies for phosphorylation or ubiquitination sites

By applying these insights from the broader field of RING finger protein research, investigators can enhance the specificity, reliability, and utility of antibodies in RNF144AA studies.

How might advances in antibody engineering enhance the study of RNF144AA membrane dynamics?

Emerging antibody technologies offer exciting possibilities for studying RNF144AA membrane dynamics:

• Single-domain antibodies and nanobodies:

  • Their small size (~15 kDa vs ~150 kDa for conventional antibodies) enables better access to membrane-embedded epitopes

  • Can be expressed intracellularly as "intrabodies" to track RNF144AA in living cells

  • May access the GXXXG interaction motif within the transmembrane domain that conventional antibodies cannot reach

• Conformation-specific antibodies:

  • Development of antibodies that specifically recognize oligomeric versus monomeric RNF144AA

  • Antibodies that distinguish between active and inactive conformations of the RING domains

  • Approaches include:

    • Phage display selections against specific conformations

    • Immunization with crosslinked oligomers versus monomeric protein

    • Selection strategies with competing antigens to enhance specificity

• Live-cell imaging applications:

  • Fluorescently labeled Fab fragments for real-time tracking of RNF144AA

  • Split-fluorescent protein complementation with nanobodies to visualize RNF144AA oligomerization

  • Fluorescence correlation spectroscopy with labeled antibody fragments to measure diffusion dynamics

• Methodological advancements:

  • Super-resolution microscopy compatible antibody probes (e.g., small fluorescent tags)

  • Proximity-dependent labeling using antibody-enzyme fusions

  • Antibody-directed chemical crosslinking to capture transient interactions

• Potential research applications:

  • Investigating RNF144AA clustering during activation

  • Mapping distribution in membrane microdomains

  • Tracking conformational changes during substrate recognition

These advanced antibody technologies will enable researchers to address fundamental questions about RNF144AA dynamics that were previously technically challenging to explore.

What are the most promising approaches for developing highly specific antibodies against the RNF144AA transmembrane domain?

Developing specific antibodies against the RNF144AA transmembrane domain presents unique challenges but offers several promising approaches:

• Synthetic peptide strategies:

  • Design peptides that include portions of the TM domain with flanking hydrophilic regions

  • Use specialized adjuvants for membrane protein immunization

  • Employ liposome or nanodisc presentation of the TM domain to maintain native conformation

  • Consider multiple species immunization to overcome tolerance to conserved domains

• Recombinant protein approaches:

  • Express the TM domain fused to carrier proteins that enhance solubility and immunogenicity

  • Use detergent-solubilized or amphipol-stabilized TM domain preparations

  • Express fragments containing the TM domain in membrane-mimetic systems

• Advanced selection technologies:

  • Phage display selections with competitive elution to enhance specificity

  • Negative selection against related RNF proteins to reduce cross-reactivity

  • Deep sequencing and computational analysis to identify binding modes specific to TM domains

  • Yeast display with conformational sensors to select conformation-specific binders

• Validation strategies:

  • Test against TM domain mutants (G252L/G256L) to verify specificity

  • Compare staining patterns between wild-type and TM domain deletion mutants

  • Use parallel antibodies targeting other domains as references

  • Employ knockout/knockdown models as definitive negative controls

• Application-specific considerations:

  • For studies of the GXXXG motif: Antibodies that differentiate between wild-type and G252L/G256L mutants

  • For oligomerization studies: Antibodies that preferentially recognize self-associated forms

  • For cancer research: Antibodies that can distinguish the cancer-associated G252D mutant form

By combining these approaches with rigorous validation protocols, researchers can develop highly specific tools for investigating the critical functions of the RNF144AA transmembrane domain in normal biology and disease states.