FAM101A Antibody

What is FAM101A and what cellular functions does it perform?

FAM101A (family with sequence similarity 101, member A) is a protein involved in regulating the perinuclear actin network and nuclear shape through its interaction with filamins. It plays an essential role in actin cytoskeleton formation in developing cartilaginous cells . The protein has a calculated molecular weight of 24 kDa (216 amino acids) but is typically observed at approximately 26 kDa in experimental conditions . FAM101A is also known by alternative names including RFLNA, FLJ44614, and CFM2 in various research literature . Understanding its role in cytoskeletal organization makes it a target of interest in developmental biology and cell structure research.

What species reactivity is available for FAM101A antibodies?

Most commercially available FAM101A antibodies demonstrate reactivity with human, mouse, and rat samples . Some antibody clones offer broader cross-species reactivity, including:

When selecting an antibody for your research, it is crucial to verify the specific reactivity claimed by the manufacturer and consider validation in your particular experimental system .

What applications are FAM101A antibodies validated for?

FAM101A antibodies are validated for multiple laboratory techniques with varying degrees of optimization:

The application-specific dilutions should be optimized for each experimental system as performance can vary based on sample type and detection method .

What are the critical considerations for FAM101A antibody validation?

Antibody validation is essential for ensuring experimental reproducibility. For FAM101A antibodies, consider implementing:

• Specificity testing: Verify target specificity using knockout/knockdown models or comparing multiple antibodies against different epitopes.

• Cross-reactivity assessment: Test for potential cross-reactivity with other proteins, especially in the face of published concerns about antibody specificity in research settings .

• Application-specific validation: An antibody performing well in Western blot may not necessarily work for immunohistochemistry or other applications.

• Reproducibility testing: Ensure consistent results across multiple experiments under identical conditions.

• Positive and negative controls: Include appropriate controls in every experiment, particularly tissue samples known to express or lack FAM101A expression .

Current literature emphasizes that commercially available research-grade antibodies often fail rigorous validation when they lack specificity or fail to recognize the intended target reliably .

What buffer systems and antigen retrieval methods are recommended for FAM101A immunohistochemistry?

For optimal FAM101A detection in immunohistochemistry applications:

• Antigen retrieval recommendations:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative option: Citrate buffer pH 6.0

• Dilution ranges:

  • Typical IHC dilution: 1:20-1:200 (Proteintech)

  • Alternative range: 1:100-1:500 (Novatein)

• Detection systems:

  • Both DAB-based chromogenic and fluorescence-based systems can be used depending on research needs

  • Multi-color immunofluorescence may be beneficial for co-localization studies with actin filaments or other cytoskeletal components

Remember that optimal conditions may vary based on sample type, fixation method, and the specific FAM101A antibody clone being used .

How should FAM101A antibodies be stored and handled to maintain reactivity?

Proper storage and handling are critical for maintaining antibody performance:

Some smaller volume preparations (20μL) may contain 0.1% BSA as a stabilizer . When handling, always wear appropriate personal protective equipment due to the presence of sodium azide and follow institutional safety guidelines.

What Western blotting protocols have been optimized for FAM101A detection?

For optimal Western blot detection of FAM101A:

• Sample preparation:

  • HepG2 cells, human skeletal muscle tissue, and mouse spleen tissue have been validated as positive sources

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors

• Protein loading and separation:

  • Load 20-30μg total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal separation around the 26 kDa range

• Transfer and detection parameters:

  • Semi-dry or wet transfer systems (PVDF membrane recommended)

  • Typical antibody dilution: 1:500-1:2000

  • Blocking: 5% non-fat milk or BSA in TBST

  • Expected band size: 26 kDa (observed) versus 24 kDa (calculated)

• Troubleshooting non-specific bands:

  • Increase antibody dilution

  • Extend washing steps

  • Consider alternative blocking reagents if background is high

Specific protocols can be obtained from manufacturers, with Proteintech offering downloadable protocols optimized for their FAM101A antibody (21113-1-AP) .

How can researchers evaluate potential cross-reactivity in FAM101A antibody applications?

Cross-reactivity remains a significant concern in antibody-based research. For FAM101A antibodies, consider these approaches:

• Multi-method validation:

  • Compare results across different techniques (WB, IHC, IF)

  • Use antibodies targeting different epitopes of FAM101A

  • Implement peptide competition assays to confirm specificity

• Control samples:

  • Include FAM101A knockout/knockdown samples when available

  • Use tissues known to have differential expression of FAM101A

  • Consider both positive and negative control cell lines

• Specificity verification:

  • Some manufacturers perform verification on protein arrays containing target protein plus 383 other non-specific proteins

  • Request validation data from manufacturers before purchase

The research community has documented cases where widely-used antibodies were later found to cross-react with unintended targets. For example, the literature highlights cases like CDK1 antibody A17, which cross-reacts with CEP152 in both Western blot and immunocytochemistry applications .

What conjugated FAM101A antibodies are available for multiplexed immunofluorescence studies?

For researchers conducting multiplexed imaging studies, several conjugated FAM101A antibodies are available:

These conjugated antibodies allow visualization of FAM101A in relation to other cellular components without the need for secondary antibodies, simplifying multiplexed experimental design .

How should researchers interpret variability in FAM101A detection across different tissue samples?

When analyzing FAM101A expression across tissues, consider these interpretation guidelines:

• Tissue-specific expression patterns:

  • FAM101A has been positively detected in human skeletal muscle tissue, stomach tissue, and hepatocirrhosis tissue

  • Mouse spleen tissue also shows positive expression

  • Expression levels may vary with developmental stage in cartilaginous tissues

• Factors affecting expression variability:

  • Cell type heterogeneity within tissues

  • Developmental stage of the tissue

  • Pathological status (normal vs. disease state)

  • Technical variables including fixation methods and antibody lots

• Quantitative approach recommendations:

  • Use digital image analysis for IHC/IF quantification

  • Include housekeeping protein controls for Western blots

  • Consider qPCR validation of protein expression findings

When comparing expression across different experimental conditions, standardized protocols and consistent antibody lots are essential for minimizing technical variability that could be misinterpreted as biological differences.

What are the implications of contradictory findings in FAM101A antibody-based research?

When researchers encounter contradictory results in FAM101A studies:

• Evaluate antibody sources:

  • Different clones may recognize different epitopes or isoforms

  • Polyclonal antibodies (which all current FAM101A antibodies appear to be) may have batch-to-batch variability

• Technical considerations:

  • Fixation methods significantly impact epitope availability

  • Antibody dilution and incubation conditions affect sensitivity and specificity

  • Detection systems have different sensitivity thresholds

• Biological considerations:

  • Post-translational modifications may affect antibody recognition

  • Protein-protein interactions could mask epitopes

  • Alternative splicing might generate variants with different antibody reactivity

The scientific literature contains examples of published papers relying on cross-reactive antibodies, emphasizing that researchers should critically evaluate both their own findings and published results. Currently, there are no alert processes or repercussions for unrectracted publications using invalidated antibodies .

How does FAM101A research connect to broader cytoskeletal biology questions?

FAM101A research extends beyond the protein itself to connect with larger questions in cell biology:

• Cytoskeletal regulation networks:

  • FAM101A interacts with filamins in regulating the perinuclear actin network

  • This interaction may influence mechanotransduction pathways from cell membrane to nucleus

• Developmental biology implications:

  • Essential role in actin cytoskeleton formation in developing cartilaginous cells

  • Potential importance in cell differentiation and tissue morphogenesis

• Disease relevance:

  • Altered cytoskeletal structure is implicated in numerous pathologies

  • FAM101A expression in hepatocirrhosis tissue suggests possible roles in liver pathology

• Research approach integration:

  • Combine antibody-based detection with genetic manipulation approaches

  • Integrate structural biology insights with functional studies

  • Consider evolutionary conservation of FAM101A across species

Understanding FAM101A within this broader context helps researchers position their work within the field and identify promising future research directions.

What are the most common technical challenges when using FAM101A antibodies?

Researchers frequently encounter these technical challenges:

• Background and non-specific binding:

  • Optimize blocking solutions (5% BSA or 5% non-fat milk)

  • Increase antibody dilution if background is high

  • Extended washing steps with agitation

  • Consider detergent concentration in wash buffers

• Inconsistent detection:

  • Standardize sample preparation methods

  • Optimize antigen retrieval for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Ensure consistent antibody handling and storage

  • Consider fresh antibody aliquots for critical experiments

• Unexpected molecular weight:

  • Expected versus observed weight discrepancy (24 kDa calculated vs. 26 kDa observed)

  • Post-translational modifications may alter migration pattern

  • Confirmation with alternative antibody clones recommended

• Batch-to-batch variability:

  • Document lot numbers for reproducibility

  • Include consistent positive controls across experiments

  • Consider testing new lots against previous lots before use in critical experiments

These methodological considerations are essential for generating reproducible data with FAM101A antibodies.

How can researchers objectively evaluate FAM101A antibody quality before experimental use?

Implement these evaluation strategies before committing to experimental studies:

• Pre-purchase assessment:

  • Review validation data provided by manufacturers

  • Evaluate publication record using the specific antibody

  • Consider antibodies validated across multiple applications

• Initial validation experiments:

  • Test antibody on positive and negative control samples

  • Compare with literature-reported expression patterns

  • Verify expected molecular weight on Western blots

  • Test dilution series to determine optimal working concentration

• Quality metrics to consider:

  • Signal-to-noise ratio in various applications

  • Lot-to-lot consistency data if available

  • Purification method (affinity-purified antibodies like those from Proteintech, Novatein, and antibodies-online offer enhanced specificity)

  • Purity level (≥95% as determined by SDS-PAGE for some commercial options)

Thorough pre-experimental validation helps avoid wasted resources on unreliable reagents and ensures data integrity.

What guidelines should be followed when publishing research using FAM101A antibodies?

To enhance reproducibility and transparency in FAM101A antibody-based research:

• Essential reporting standards:

  • Complete antibody identification (manufacturer, catalog number, lot number, RRID)

  • Detailed experimental protocols including dilutions, incubation times, and detection methods

  • Description of all controls used for specificity validation

  • Representative images of both positive and negative results

• Validation documentation:

  • Include supplementary data demonstrating antibody specificity

  • Describe any validation experiments performed

  • Acknowledge limitations in antibody performance or specificity

• Addressing conflicting results:

  • Discuss contradictions with published literature

  • Consider multiple antibodies when conflicting results emerge

  • Validate findings using orthogonal techniques (e.g., mRNA expression, tagged protein expression)