FAM71A Antibody

What is FAM71A and what are its known functions?

FAM71A (family with sequence similarity 71, member A) is a protein that interacts with the Rab2B small GTPase and plays a critical role in maintaining Golgi body integrity. Research has shown that knockdown of the FAM71A gene induces fragmentation of the Golgi, similar to effects observed with Rab2B small GTPase knockdown. The protein contains an N-terminal Rab-binding domain that is specific for Rab2B, suggesting its involvement in Golgi-related cellular processes . FAM71A is also known by several aliases including GARI-L4, Golgi-associated RAB2 interactor protein 4, and testis secretory sperm-binding protein Li 209a, indicating potential roles in reproductive biology .

What are the molecular characteristics of human FAM71A?

Human FAM71A is encoded by a gene located on chromosome 1q32.3 . The protein has a molecular mass of approximately 63.177 kDa and belongs to the FAM71 protein family . The protein's UniProt accession number is Q8IYT1, and its Entrez Gene ID is 149647 . While there is still limited information on the complete structure-function relationship of FAM71A, its interaction with Rab2B suggests involvement in vesicular trafficking pathways, particularly those associated with Golgi apparatus function .

What types of FAM71A antibodies are available for research?

Multiple types of FAM71A antibodies are available for research applications, with most being polyclonal antibodies derived from rabbit hosts . These antibodies are designed to target different epitopes of the FAM71A protein, including C-terminal regions and specific immunogen sequences such as "SMSLSREGSVSLAIAGVVLTSRTAAEADMDAAAGPPVSTRQSKSSLSGQHGRERTQASAEGCKEGRERREKDRALGRSSHRRRTGESRHK" . The antibodies vary in their specific applications, with most being validated for techniques including immunohistochemistry (IHC), western blotting (WB), and immunofluorescence (ICC-IF) .

How should I optimize FAM71A antibody dilutions for immunohistochemistry?

For optimal immunohistochemistry results with FAM71A antibodies, begin with the manufacturer's recommended dilution range of 1:500-1:1000 . Consider performing a dilution series (e.g., 1:250, 1:500, 1:1000, 1:2000) to determine the optimal antibody concentration for your specific tissue samples. The sensitivity of detection systems varies, so antibody dilutions should be adjusted accordingly when switching between detection methods (e.g., from HRP-DAB to fluorescence-based detection).

For antigen retrieval, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is generally recommended, but EDTA buffer (pH 9.0) may provide better results for certain fixation conditions. Always include positive and negative controls to validate staining specificity, particularly tissue samples known to express or not express FAM71A based on Human Protein Atlas data .

What are the recommended protocols for using FAM71A antibodies in Western blot applications?

For Western blot applications using FAM71A antibodies, the following methodological approach is recommended:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. For Golgi-associated proteins like FAM71A, consider subcellular fractionation to enrich for Golgi membranes.

• Gel electrophoresis: Load 20-50 μg of total protein per lane on 10% SDS-PAGE gels, as FAM71A has a molecular weight of approximately 63 kDa .

• Transfer: Use wet transfer at 30V overnight at 4°C for optimal transfer efficiency of mid-to-large sized proteins.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute FAM71A antibody at 1:500 to 1:1000 in blocking buffer and incubate overnight at 4°C.

• Detection: Use HRP-conjugated secondary antibodies and ECL detection systems, with careful exposure time optimization to avoid background.

Remember that FAM71A may show tissue-specific expression patterns, with potential detection challenges in samples with low expression levels.

How can I validate the specificity of FAM71A antibodies?

Validating FAM71A antibody specificity requires a multi-faceted approach:

• Knockout/knockdown validation: Compare staining patterns between wild-type samples and those where FAM71A has been knocked down or knocked out using siRNA or CRISPR-Cas9 technologies. A specific antibody will show significantly reduced signal in knockdown/knockout samples.

• Overexpression validation: Transfect cells with FAM71A expression vectors and confirm increased signal intensity compared to non-transfected controls.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific binding should be blocked, resulting in signal reduction.

• Cross-reactivity assessment: Test the antibody against orthologous proteins. Some FAM71A antibodies show varying degrees of cross-reactivity with mouse (38%) and rat (38%) orthologs , while others demonstrate higher homology with cow (92%), dog (91%), guinea pig (85%), pig (100%), and rabbit (100%) .

• Orthogonal validation: Correlate protein detection with RNA expression data to ensure concordance between transcriptional and translational expression patterns.

How does FAM71A knockdown affect Golgi morphology and function in different cell types?

FAM71A knockdown has been demonstrated to induce fragmentation of the Golgi apparatus, similar to the phenotype observed with Rab2B knockdown . This suggests a functional relationship between these two proteins in maintaining Golgi structural integrity. The effects of FAM71A knockdown may vary across different cell types based on their intrinsic Golgi architecture and protein trafficking requirements.

In epithelial cells, FAM71A knockdown typically results in dispersed Golgi mini-stacks throughout the cytoplasm, while in fibroblasts, the effects may manifest as more subtle structural alterations. The functional consequences of this fragmentation include potential disruptions in protein glycosylation, sorting, and secretion. Researchers investigating this phenomenon should employ both morphological analysis (using Golgi markers like GM130, TGN46) and functional assays (protein secretion, glycosylation status assessment) to comprehensively characterize the impact of FAM71A depletion.

When designing FAM71A knockdown experiments, consider using multiple siRNA sequences to control for off-target effects, and rescue experiments with siRNA-resistant FAM71A constructs to confirm specificity.

What is known about the interaction between FAM71A and Rab2B in regulating vesicular trafficking?

The interaction between FAM71A and Rab2B represents a significant regulatory mechanism in vesicular trafficking, particularly at the ER-Golgi interface. FAM71A contains an N-terminal Rab-binding domain that specifically interacts with Rab2B , suggesting a selective role in Rab2B-dependent trafficking pathways. This interaction is likely nucleotide-dependent, with FAM71A preferentially binding to GTP-bound (active) Rab2B.

The functional consequences of this interaction may include:

• Regulation of COPI vesicle formation and retrograde trafficking

• Maintenance of Golgi cisternal organization

• Coordination of membrane tethering events preceding fusion

• Recruitment of effector proteins to specific Golgi membrane domains

To study this interaction, researchers should consider employing:

• Co-immunoprecipitation assays with GTP-locked and GDP-locked Rab2B mutants

• Proximity ligation assays to visualize interactions in situ

• FRET-based approaches to monitor dynamic interaction events

• Reconstitution assays using purified components to define minimal interaction requirements

Disruption of the FAM71A-Rab2B interaction through targeted mutations can provide insight into the specific structural determinants of this interaction and their functional significance.

What are the tissue-specific expression patterns of FAM71A and their implications for research?

Understanding the tissue-specific expression patterns of FAM71A is crucial for designing relevant experimental models and interpreting research findings. Based on the Human Protein Atlas data referenced in antibody validation studies, FAM71A shows variable expression across different tissues and cell types .

• Selecting appropriate cell lines that endogenously express FAM71A

• Understanding baseline expression levels to interpret knockdown/overexpression effects

• Recognizing potential tissue-specific interaction partners that may modify FAM71A function

• Considering developmental regulation of FAM71A expression when using developmental models

Researchers investigating FAM71A should validate its expression in their specific experimental systems using both protein detection methods (immunohistochemistry, western blotting) and transcript analysis (qRT-PCR, RNA-seq) to ensure the relevance of their model system.

How can I resolve non-specific binding issues when using FAM71A antibodies?

Non-specific binding is a common challenge when working with FAM71A antibodies. To resolve this issue, implement the following strategies:

• Optimize blocking conditions by testing different blocking agents (BSA, normal serum, commercial blocking reagents) and concentrations (3-5%).

• Increase washing stringency by using higher detergent concentrations (0.1-0.3% Tween-20 or Triton X-100) and extending wash durations.

• Titrate antibody concentrations carefully, as over-dilution can reduce specific signal while under-dilution increases background.

• For immunohistochemistry and immunofluorescence, pre-absorb antibodies with tissue homogenates from species matching your sample but lacking the target protein.

• Consider alternative detection systems - some FAM71A antibodies may perform better with certain detection methods (e.g., biotin-streptavidin amplification versus direct HRP conjugation).

• For western blotting, extended membrane blocking (overnight at 4°C) and antibody dilution in alternative buffers (such as 5% BSA instead of milk) can reduce non-specific binding to certain proteins.

If non-specific binding persists, consider validating alternative FAM71A antibodies targeting different epitopes, as some regions of the protein may be more prone to non-specific interactions.

What are the potential cross-reactivity considerations when using FAM71A antibodies in different species?

Cross-reactivity considerations are essential when applying FAM71A antibodies across different species. Based on sequence homology data, different FAM71A antibodies demonstrate varying degrees of cross-reactivity:

When planning cross-species applications, consider:

• Epitope conservation analysis: Align the immunogen sequence with the corresponding region in your species of interest to predict potential binding.

• Pilot validation: Always perform initial validation in your species of interest, even if the manufacturer claims cross-reactivity.

• Controlled comparison: Include human samples (if available) as positive controls to benchmark detection sensitivity and specificity in your experimental species.

• Alternative antibody selection: If cross-reactivity is poor, consider antibodies raised against more conserved regions of FAM71A or species-specific antibodies.

• Epitope retrieval optimization: Cross-reactive antibodies may require modified epitope retrieval methods in different species due to fixation-related differences in epitope accessibility.

What controls should be included when designing experiments using FAM71A antibodies?

Rigorous experimental design with appropriate controls is crucial for generating reliable data with FAM71A antibodies:

• Positive tissue/cell controls:

  • Include samples known to express FAM71A based on literature or database information

  • Consider using cell lines with validated FAM71A expression

• Negative controls:

  • Samples from FAM71A knockout models

  • Tissues known to lack FAM71A expression

  • Primary antibody omission controls

  • Isotype controls matched to the FAM71A antibody's host species and isotype

• Expression manipulation controls:

  • siRNA/shRNA knockdown samples to demonstrate specificity

  • Overexpression samples showing increased signal intensity

• Peptide competition controls:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Use unrelated peptides as negative controls for peptide competition

• Technical controls:

  • Include loading controls (e.g., GAPDH, actin) for western blots

  • Use housekeeping gene detection in parallel for normalization

  • Process all experimental groups simultaneously to minimize technical variability

Documentation of control results alongside experimental findings is essential for proper interpretation and should be included in all research publications.

How should researchers interpret FAM71A localization patterns in relation to Golgi markers?

When interpreting FAM71A localization patterns, researchers should consider its relationship with established Golgi markers. FAM71A typically shows partial co-localization with Golgi markers, reflecting its specialized role in Golgi-related functions . For precise interpretation:

Remember that fixation and permeabilization methods can significantly affect the preservation and accessibility of Golgi structures, potentially altering apparent localization patterns.

What experimental approaches can be used to study the functional consequences of FAM71A mutations?

To study the functional consequences of FAM71A mutations, researchers can employ multiple complementary approaches:

• CRISPR-Cas9 genome editing:

  • Generate point mutations in endogenous FAM71A

  • Create domain-specific deletions

  • Establish complete knockout cell lines as controls

• Structure-function analysis:

  • Express truncation mutants to identify functional domains

  • Create chimeric proteins to assess domain-specific activities

  • Perform alanine scanning mutagenesis of key residues within the Rab-binding domain

• Functional readouts:

  • Assess Golgi morphology using immunofluorescence microscopy

  • Measure protein secretion rates of model cargo proteins

  • Analyze glycosylation patterns of secreted proteins

  • Evaluate trafficking kinetics using live-cell imaging of fluorescent cargo

• Interaction studies:

  • Perform co-immunoprecipitation with Rab2B and other potential partners

  • Use proximity-based methods (BioID, APEX) to identify near-neighbors

  • Conduct in vitro binding assays with purified components

• Physiological consequences:

  • Analyze cell growth, division, and migration

  • Assess tissue-specific functions in relevant cell types

  • Examine developmental effects in model organisms

When interpreting results, consider that mutations affecting the Rab2B-binding domain are likely to have similar phenotypes to FAM71A knockdown, while mutations in other regions might reveal additional functions beyond Golgi maintenance.

How can FAM71A research contribute to understanding diseases associated with Golgi dysfunction?

FAM71A research has significant potential to advance our understanding of diseases associated with Golgi dysfunction, given its role in maintaining Golgi integrity through interaction with Rab2B . Several opportunities exist for translational research:

• Neurodegenerative disorders:

  • Many neurodegenerative diseases involve disrupted protein trafficking

  • FAM71A dysfunction could contribute to pathogenic protein accumulation

  • Investigating FAM71A in neuronal models of Alzheimer's, Parkinson's, or ALS could reveal disease-relevant mechanisms

• Congenital disorders of glycosylation (CDGs):

  • Golgi dysfunction leads to aberrant glycosylation patterns

  • FAM71A's role in maintaining Golgi structure may impact glycosylation efficiency

  • Compare FAM71A expression and function in CDG patient-derived cells versus controls

• Cancer biology:

  • Altered secretory pathway function supports cancer cell migration and invasion

  • Examine FAM71A expression across cancer types and correlate with invasive phenotypes

  • Investigate whether targeting FAM71A-Rab2B interaction affects cancer cell biology

• Inflammatory and autoimmune conditions:

  • Secretion of inflammatory mediators depends on Golgi function

  • Study how FAM71A affects cytokine secretion in immune cells

  • Examine whether FAM71A expression changes during inflammatory responses

• Therapeutic development:

  • The FAM71A-Rab2B interaction interface represents a potential drug target

  • Screening for small molecules that modulate this interaction could yield therapeutic leads

  • Peptide-based inhibitors mimicking interaction domains could serve as research tools

Researchers should consider interdisciplinary collaborations with clinicians to obtain relevant patient samples for validating FAM71A findings in disease contexts.

What are the emerging research directions for FAM71A?

The study of FAM71A is still in relatively early stages, with several promising research directions emerging. As understanding of this protein's function in Golgi maintenance continues to develop, researchers should consider exploring:

• Structural biology approaches to determine the three-dimensional structure of FAM71A, particularly its Rab2B-binding domain, to understand the molecular basis of this critical interaction.

• Comprehensive interactome mapping using proximity labeling techniques (BioID, APEX) combined with mass spectrometry to identify the full spectrum of FAM71A protein interactions beyond Rab2B.

• Tissue-specific functions of FAM71A, especially in testis where aliases suggest potential specialized roles in reproductive biology .

• Regulatory mechanisms controlling FAM71A expression, localization, and function, including potential post-translational modifications and their functional consequences.

• Development of tools targeting FAM71A function, such as cell-permeable peptides disrupting specific protein interactions or small molecule modulators of its activity.

• Examination of FAM71A homologs and their comparative functions across species to understand evolutionary conservation and divergence of FAM71 family proteins.