FAM127A Antibody

What is FAM127A and what are its alternative names in scientific literature?

FAM127A (family with sequence similarity 127, member A) is also known by several alternative names in scientific literature, including RTL8C, CXX1, MAR8C, MART8C, Mar8, Mart8, SIRH5, and CAAX box protein 1. These nomenclature variations should be considered when conducting literature searches to ensure comprehensive coverage of relevant research . The protein has a calculated molecular mass of approximately 22 kDa, though it is typically observed at around 40 kDa in Western blot applications, potentially indicating post-translational modifications or alternative splicing variants .

What are the key characteristics of FAM127A antibodies currently available for research?

Current commercially available FAM127A antibodies are primarily polyclonal antibodies raised in rabbits. They are typically generated using recombinant protein immunogens representing specific amino acid sequences of human FAM127A. For instance, some antibodies target a sequence corresponding to amino acids 1-113 of human FAM127A (NP_001071639.1) . The antibodies demonstrate reactivity primarily with human samples, though cross-reactivity with mouse and rat samples has been reported for certain antibodies . These antibodies are commonly supplied in PBS buffer containing sodium azide and glycerol at pH 7.3 and should be stored at -20°C to maintain optimal activity .

How do the different FAM127 family members compare structurally, and what implications does this have for antibody specificity?

The FAM127 family includes highly homologous proteins that share significant sequence similarity. Some antibody immunogens have over 95% sequence similarity among family members , which creates potential cross-reactivity challenges. When selecting a FAM127A-specific antibody, researchers should examine the exact immunogen sequence used and validate specificity through appropriate controls. The high homology among family members (all composed of 113 amino acids with extremely high sequence similarity ) means that antibodies may recognize multiple FAM127 proteins unless specifically engineered for unique epitopes. This structural similarity necessitates careful experimental design and validation when specific family member detection is required.

What are the validated applications for FAM127A antibodies and their recommended dilution ranges?

FAM127A antibodies have been validated for multiple experimental applications:

For optimal results, antibody dilution should be empirically determined for each specific experimental system. For IHC applications with FAM127A antibodies, antigen retrieval using TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 can also be effective . For Western blot applications, secondary detection typically employs HRP-conjugated anti-rabbit IgG antibodies .

What are the effective protocols for detecting nuclear localization of FAM127A using immunofluorescence?

For immunofluorescence detection of nuclear-localized FAM127A:

• Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 (5-10 minutes)

• Block with 5% normal serum in PBS with 0.1% Tween-20 (1 hour)

• Incubate with primary anti-FAM127A antibody (1:25-1:100 dilution, overnight at 4°C)

• Wash 3x with PBS-T

• Incubate with fluorophore-conjugated secondary antibody (e.g., Rhodamine-labeled anti-rabbit IgG, 1 hour at room temperature)

• Counterstain nuclei with DAPI

• Mount and image

When examining nuclear localization, co-staining with markers of subnuclear structures such as NPM1 (for nucleoli) or PML (for PML bodies) is recommended to precisely determine the subnuclear compartmentalization of FAM127A . Evidence suggests that FAM127A/RTL8 localizes to nucleoli, making this co-staining approach particularly informative .

How should researchers optimize Western blot protocols for detecting endogenous versus overexpressed FAM127A?

For optimal Western blot detection of FAM127A:

Endogenous FAM127A detection:

• Prepare lysates in RIPA buffer with protease inhibitors

• Load 25-40 μg total protein per lane

• Use 12-15% SDS-PAGE gels to resolve the approximately 40 kDa band

• Transfer to PVDF membrane at 100V for 60-90 minutes

• Block with 3-5% nonfat dry milk in TBST (1 hour at room temperature)

• Incubate with anti-FAM127A antibody at 1:500-1:1000 dilution (overnight at 4°C)

• Use HRP-conjugated secondary antibody at 1:10,000 dilution

• Develop with ECL substrate with appropriate exposure time (typically 1-60 seconds)

Overexpressed FAM127A detection:

• Reduce primary antibody concentration to 1:1000-1:3000 to prevent oversaturation

• Reduce protein loading to 10-20 μg per lane

• Consider using epitope tags (HA, FLAG) for specific detection of overexpressed protein

• Include untransfected controls to distinguish endogenous from overexpressed protein

The observed molecular weight of FAM127A in Western blots (approximately 40 kDa) differs from the calculated mass (22 kDa), so researchers should be aware of this discrepancy when interpreting results .

How can researchers effectively study the interaction between FAM127A/RTL8 and UBQLN2 in nuclear translocation experiments?

To study FAM127A/RTL8-mediated nuclear translocation of UBQLN2:

• Co-transfect cells (e.g., HEK293) with tagged constructs (FLAG-UBQLN2 and HA-RTL8)

• Perform immunofluorescence staining at 24-48 hours post-transfection

• Include single-transfection controls to establish baseline localization patterns

• Quantify nuclear/cytoplasmic distribution ratios of UBQLN2 in the presence/absence of RTL8

• Employ confocal microscopy with Z-stack imaging to accurately assess co-localization in nuclear puncta

What are the challenges in distinguishing between FAM127A and related family members in experimental settings?

Due to the high sequence homology among FAM127 family members, researchers face several challenges:

• Antibody cross-reactivity: Most available antibodies may recognize multiple FAM127 family members due to their high sequence similarity (>95% in some regions)

• Transcript specificity: Design gene-specific primers targeting unique regions for RT-PCR or qPCR

• Protein detection specificity: Consider using:

  • Epitope-tagged constructs for overexpression studies

  • RNA interference with validated siRNAs targeting specific family members

  • CRISPR-Cas9 gene editing with careful validation of knockout specificity

To overcome these challenges, researchers should:

• Perform careful antibody validation using positive and negative controls

• Include parallel experiments with known FAM127A-expressing and non-expressing cell lines

• Consider using multiple detection methods to corroborate findings

• When possible, use mass spectrometry for unambiguous protein identification

How can researchers investigate the role of FAM127A in nucleolar function and protein quality control pathways?

To investigate FAM127A's role in nucleolar function and protein quality control:

• Co-localization studies:

  • Perform immunofluorescence with antibodies against nucleolar markers (NPM1) and FAM127A

  • Use super-resolution microscopy techniques (STED, STORM) for precise spatial mapping

• Functional studies:

  • Employ CRISPR-Cas9 to generate FAM127A knockout cells

  • Use inducible expression systems to control FAM127A levels

  • Analyze nucleolar morphology, composition, and function in response to FAM127A manipulation

• Protein quality control analysis:

  • Examine co-localization with markers of protein quality control (Hsp70, p62, ubiquitin)

  • Assess protein ubiquitination and degradation rates in presence/absence of FAM127A

  • Employ proteasome inhibitors to determine FAM127A's role in protein degradation pathways

Research indicates that RTL8/FAM127A localizes to nucleoli exclusively, while overexpressed RTL8A concentrates in proximity to PML bodies. Additionally, RTL8A puncta co-localize with Hsp70, p62, and ubiquitin, suggesting involvement in protein quality control mechanisms . These experimental approaches can help elucidate FAM127A's role in these critical cellular processes.

What are common issues encountered in FAM127A Western blot experiments and how can they be resolved?

When interpreting FAM127A Western blot results, remember that the observed molecular weight (~40 kDa) differs significantly from the calculated weight (~22 kDa) . This discrepancy should be considered when assessing specificity and may reflect important biological properties of the protein rather than technical artifacts.

How can researchers optimize immunohistochemical detection of FAM127A in different tissue types?

For optimal IHC detection of FAM127A across different tissues:

• Tissue-specific optimization:

  • Human pancreatic cancer tissue: Use TE buffer (pH 9.0) for antigen retrieval

  • Human colon tissue: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Brain tissue: May require extended antigen retrieval times (20-30 minutes)

• Signal enhancement strategies:

  • Consider tyramide signal amplification for low-abundance detection

  • Optimize primary antibody incubation time (overnight at 4°C often yields best results)

  • Test different detection systems (ABC, polymer-based) to determine optimal sensitivity

• Control protocols:

  • Include positive control tissues (human pancreas, colon, brain)

  • Perform peptide competition assays to validate specificity

  • Include isotype control antibodies at equivalent concentrations

The recommended dilution range for FAM127A antibodies in IHC applications is 1:20-1:200, but this should be empirically determined for each tissue type and fixation method . Variations in fixation time, processing, and storage can significantly impact antigen preservation and detection sensitivity.

What are the critical considerations for designing experiments to study FAM127A's role in protein quality control and nuclear trafficking?

When designing experiments to investigate FAM127A's role in protein quality control and nuclear trafficking:

• Selection of cellular stress models:

  • Heat shock (42°C, 1-2 hours)

  • Proteasome inhibition (MG132, 5-10 μM, 4-16 hours)

  • Oxidative stress (H₂O₂, 100-500 μM, 1-4 hours)

• Temporal dynamics:

  • Perform time course experiments to capture dynamic relocalization

  • Use live-cell imaging with fluorescently tagged FAM127A constructs

  • Consider pulse-chase experiments to track protein fate

• Interaction partners:

  • Design co-immunoprecipitation experiments to identify FAM127A-interacting proteins

  • Include appropriate controls (IgG, reverse IP)

  • Consider proximity labeling approaches (BioID, APEX) to identify transient interactions

• Functional readouts:

  • Measure protein aggregation and clearance in the presence/absence of FAM127A

  • Assess ubiquitination status of potential substrates

  • Quantify nuclear-cytoplasmic distribution of interaction partners like UBQLN2

Research indicates that FAM127A/RTL8 promotes the nuclear localization of UBQLN2 to subnuclear compartments, and the RTL8-UBQLN2 interaction appears to play a role in protein quality control pathways involving Hsp70, p62, and ubiquitin . These considerations will help researchers design robust experiments to further elucidate these complex molecular mechanisms.

How can new methodologies enhance the study of FAM127A's nuclear functions and interactions?

Emerging methodologies that can advance FAM127A research include:

• Proximity-based interaction mapping:

  • BioID or TurboID fusion proteins to identify proteins in close proximity to FAM127A

  • APEX2-mediated biotinylation for temporal control of interaction mapping

  • Split protein complementation assays to visualize direct interactions in living cells

• Advanced imaging approaches:

  • Super-resolution microscopy (STED, STORM, PALM) for precise subnuclear localization

  • Lattice light-sheet microscopy for extended live-cell imaging with reduced phototoxicity

  • Single-molecule tracking to follow FAM127A dynamics within nuclear compartments

• Genomic and transcriptomic integration:

  • CUT&RUN or CUT&Tag approaches to identify chromatin associations

  • RNA-protein interaction mapping (CLIP-seq variants) if RNA-binding functions are suspected

  • Spatial transcriptomics to correlate FAM127A localization with gene expression patterns

These methodologies would be particularly valuable for studying the nucleolar localization of endogenous RTL8/FAM127A and its potential roles in nuclear protein quality control pathways, as suggested by research showing its co-localization with nucleolar markers and protein quality control components .

What is the significance of FAM127A's association with nucleoli and PML bodies, and how can this be further investigated?

The association of FAM127A/RTL8 with nucleoli and PML bodies suggests potential roles in:

• Ribosome biogenesis regulation:

  • Investigate FAM127A's impact on pre-rRNA processing using pulse-chase labeling

  • Perform nucleolar proteomics in FAM127A-depleted cells

  • Assess ribosome assembly and function in FAM127A knockout models

• Nuclear protein quality control:

  • Examine FAM127A's role in degradation of misfolded nuclear proteins

  • Study its function during proteotoxic stress conditions

  • Investigate potential roles in nucleolar stress responses

• PML body functions:

  • Analyze FAM127A's contribution to PML body formation and composition

  • Examine its role in cellular responses to viral infection and oxidative stress

  • Investigate potential participation in selective autophagy of nuclear components

Research has shown that endogenous RTL8/FAM127A localizes exclusively to nucleoli, while overexpressed RTL8A concentrates near PML bodies . Further investigation of these distinct localization patterns could reveal context-dependent functions of FAM127A in different subnuclear compartments and their associated cellular processes.

How do the interactions between FAM127A, UBQLN2, and protein quality control pathways inform potential therapeutic approaches?

The emerging understanding of FAM127A's role in protein quality control pathways suggests several therapeutic implications:

• Neurodegenerative disease relevance:

  • UBQLN2 has been implicated in ALS/FTD and Huntington's disease

  • FAM127A's role in UBQLN2 nuclear localization may influence disease progression

  • Investigate FAM127A expression in neurodegenerative disease models and patient samples

• Cancer biology connections:

  • Assess FAM127A expression in various cancer types and correlation with outcomes

  • Determine if FAM127A-mediated protein quality control influences tumor cell survival

  • Evaluate potential as a therapeutic target or biomarker

• Therapeutic modulation strategies:

  • Develop small molecule inhibitors of FAM127A-UBQLN2 interaction

  • Explore peptide-based approaches to disrupt specific protein-protein interactions

  • Consider antisense oligonucleotides for selective modulation of FAM127A expression

Research indicating that FAM127A/RTL8 promotes nuclear localization of UBQLN2 and that UBQLN2 can facilitate clearance of mutant Huntingtin protein suggests potential relevance to neurodegenerative disease mechanisms . Further investigation of these pathways could reveal novel therapeutic approaches for conditions involving aberrant protein aggregation and nuclear protein quality control.