FAM120A Antibody

What is FAM120A and what cellular functions does it participate in?

FAM120A (family with sequence similarity 120A) is a protein with a calculated molecular weight of 122 kDa (1118 amino acids), though it is typically observed at approximately 125 kDa in experimental contexts . Also known as OSSA (Oxidative stress-associated Src activator), this protein has been identified as a constitutive coactivator of PPAR-gamma-like protein and DNA polymerase-transactivated protein . FAM120A has been studied in relation to multiple cellular processes, particularly those involving oxidative stress responses. It is encoded by the gene with NCBI ID 23196 and has the UniProt ID Q9NZB2 . The protein is widely expressed across human tissues, with notable cytoplasmic localization in glandular cells as demonstrated by immunohistochemical analysis of human colon samples .

What applications can FAM120A antibodies be reliably used for?

FAM120A antibodies have been validated for multiple research applications:

Researchers should note that optimal dilutions may vary depending on sample type and experimental conditions, making it essential to titrate the antibody in each specific testing system to obtain optimal results .

What is the species reactivity of commercial FAM120A antibodies?

Commercial FAM120A antibodies demonstrate cross-reactivity with samples from multiple mammalian species:

When selecting an antibody for cross-species applications, researchers should review validation data for their species of interest, as reactivity may vary between antibody clones and manufacturers .

What are the recommended storage conditions for FAM120A antibodies?

Proper storage is critical for maintaining antibody activity over time. For FAM120A antibodies:

• Store at -20°C for long-term preservation

• Most formulations remain stable for one year after shipment when stored properly

• Antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• For smaller size aliquots (20 μl), solutions may contain 0.1% BSA as a stabilizer

• Aliquoting is generally unnecessary for -20°C storage with the glycerol-containing buffer formulations

Researchers should avoid repeated freeze-thaw cycles to maintain antibody performance over time, especially for formulations without glycerol protection .

How can I validate the specificity of FAM120A antibody in my experimental system?

Validating antibody specificity is essential for generating reliable research data. For FAM120A antibodies, consider implementing these validation approaches:

• siRNA knockdown validation: Compare Western blot results between control samples and samples treated with siRNA targeting FAM120A. Proteintech's antibody (21529-1-AP) has been validated using this approach, demonstrating significant signal reduction in siRNA-FAM120A treated samples compared to siRNA controls .

• Molecular weight verification: Confirm that your detected band appears at the expected molecular weight of approximately 125 kDa, which matches the observed molecular weight for FAM120A .

• Positive control samples: Include validated positive control samples in your experiments. HEK-293 cells are recommended positive controls for Western blot applications with FAM120A antibodies .

• Multiple detection methods: Validate findings using complementary techniques (e.g., validate Western blot findings with immunofluorescence or immunohistochemistry) to confirm consistent expression patterns across methodologies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application to samples; specific signals should be significantly reduced or eliminated.

What antigen retrieval methods are recommended for FAM120A immunohistochemistry?

Effective antigen retrieval is critical for optimal immunohistochemical detection of FAM120A in formalin-fixed, paraffin-embedded (FFPE) tissues:

• Primary recommendation: TE buffer at pH 9.0 is suggested as the optimal antigen retrieval solution for FAM120A detection in paraffin-embedded tissues .

• Alternative method: Citrate buffer at pH 6.0 can be used as an alternative approach if the primary method yields suboptimal results .

• For NBP1-86715 antibody: HIER (Heat-Induced Epitope Retrieval) at pH 6.0 is specifically recommended for this antibody formulation .

• Protocol considerations: The complete IHC protocol for FAM120A detection should include paraffin removal, rehydration, antigen retrieval, blocking, primary antibody incubation (at dilutions between 1:20-1:200), followed by appropriate detection systems .

The choice between high pH (pH 9.0) and low pH (pH 6.0) retrieval solutions should be empirically determined for each tissue type, as fixation conditions and tissue characteristics can influence retrieval efficiency.

How can I troubleshoot weak or inconsistent Western blot signals with FAM120A antibody?

When facing challenges with Western blot detection of FAM120A, consider these methodological adjustments:

• Optimize protein loading: FAM120A may have variable expression levels across tissues and cell lines. For initial optimization, load 25-50 μg of total protein lysate.

• Adjust antibody concentration: If signals are weak, try increasing the antibody concentration gradually. The recommended dilution range for Western blot is 1:500-1:2000, but this may need adjustment based on your specific samples .

• Extend incubation time: Consider extending primary antibody incubation to overnight at 4°C rather than shorter incubations at room temperature. The validation data shows successful detection with 1:500 dilution incubated at room temperature for 1.5 hours .

• Sample preparation optimization: Ensure complete protein denaturation and use fresh protease inhibitors during lysis. For membrane-associated proteins like FAM120A, consider using lysis buffers containing mild detergents.

• Transfer efficiency verification: Use reversible total protein stains (Ponceau S) to confirm successful transfer before antibody incubation.

• Positive controls: Include HEK-293 cell lysate as a positive control, as this has been validated for FAM120A detection .

• Signal enhancement methods: For particularly challenging samples, consider using signal enhancement systems compatible with your detection method (chemiluminescence enhancers for HRP systems, for example).

What cellular localization pattern should I expect when using FAM120A antibody in immunofluorescence?

When conducting immunofluorescence experiments with FAM120A antibodies, the expected subcellular localization pattern provides important information for result interpretation:

• Primary localization: FAM120A predominantly displays cytoplasmic localization in most cell types. Immunocytochemistry/immunofluorescence studies in human A-431 cells demonstrate positive staining specifically in the cytoplasm .

• Cell line considerations: Validated cell lines for IF/ICC applications include HepG2 cells (recommended in Proteintech's antibody datasheet) and A-431 cells (validated with Novus Biologicals' antibody) .

• Fixation and permeabilization: For optimal results, PFA fixation followed by Triton X-100 permeabilization is recommended for FAM120A immunofluorescence detection .

• Dilution optimization: Start with a dilution in the middle of the recommended range (1:50-1:500), then adjust based on signal intensity .

• Tissue-specific patterns: In tissue sections, FAM120A shows strong cytoplasmic positivity specifically in glandular cells, as demonstrated in human colon samples . This pattern provides a useful reference for validating antibody performance in tissue immunofluorescence applications.

How can I optimize FAM120A antibody for co-immunoprecipitation experiments?

While co-immunoprecipitation (Co-IP) is not explicitly listed among the validated applications for the FAM120A antibodies in the search results, researchers can adapt these antibodies for Co-IP by following these methodological approaches:

• Antibody selection: Choose antibodies that have been affinity-purified, such as the antigen affinity-purified polyclonal antibodies described in the search results . These antibodies have undergone purification steps that reduce non-specific binding.

• Cross-linking consideration: For studies investigating FAM120A protein complexes, consider using reversible cross-linking agents to stabilize transient interactions before cell lysis.

• Lysis buffer optimization: Use mild lysis conditions to preserve protein-protein interactions. A buffer containing 150 mM NaCl, 1% NP-40 or Triton X-100, 50 mM Tris pH 7.5, with protease and phosphatase inhibitors is a reasonable starting point.

• Antibody titration: Begin with 2-5 μg of antibody per 500 μg of total protein lysate, adjusting based on preliminary results.

• Pre-clearing lysates: Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding.

• Controls: Include critical controls such as:

  • IgG control (same species as the FAM120A antibody)

  • Input samples (typically 5-10% of the lysate used for IP)

  • If possible, a FAM120A-depleted sample as a negative control

• Elution conditions: Optimize elution conditions based on the detection method; for Western blot analysis, standard SDS sample buffer at 95°C is typically sufficient.

How should I select between polyclonal and monoclonal antibodies for FAM120A research?

When designing experiments to study FAM120A, the choice between polyclonal and monoclonal antibodies represents an important consideration:

The search results primarily describe polyclonal rabbit antibodies against FAM120A . These polyclonal antibodies offer certain advantages for research applications:

• Epitope recognition: Polyclonal antibodies recognize multiple epitopes on the FAM120A protein, potentially providing higher sensitivity, especially in applications where protein conformations may be altered (like formalin-fixed tissues).

• Cross-species reactivity: The polyclonal antibodies described show reactivity across multiple species (human, mouse, rat), making them versatile tools for comparative studies .

• Application versatility: The described antibodies have been validated across multiple applications (WB, IHC, IF/ICC, ELISA), demonstrating their flexibility .

• Lot-to-lot variability: Polyclonal antibodies may show greater variability between production lots compared to monoclonals.

• Background concerns: In some applications, polyclonal antibodies may produce higher background than monoclonals, requiring careful optimization of blocking and washing steps.

For research requiring the highest specificity for a single epitope, researchers might consider investigating whether monoclonal options are available beyond those described in the search results.

What controls should be included when publishing research using FAM120A antibodies?

Publication-quality research using FAM120A antibodies should include these critical controls:

• Positive controls: Include validated positive control samples:

  • For Western blot: HEK-293 cells

  • For IHC: Human skeletal muscle tissue

  • For IF/ICC: HepG2 or A-431 cells

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at matching concentration)

  • Where possible, FAM120A-knockdown or knockout samples

• Validation across methods: When making substantive claims about FAM120A expression or function, validate findings using at least two independent techniques (e.g., WB and IF, or IHC and qPCR).

• Antibody reporting: Include complete antibody details in publications:

  • Manufacturer and catalog number

  • Host species and clonality

  • RRID (Research Resource Identifier) when available (e.g., AB_10860261)

  • Dilution used

  • Incubation conditions

These controls not only strengthen the validity of research findings but also facilitate reproducibility by other laboratories.

How can I differentiate between specific and non-specific signals when using FAM120A antibodies?

Distinguishing genuine FAM120A signals from artifacts requires careful analysis and validation:

• Molecular weight verification: FAM120A has a calculated molecular weight of 122 kDa, with observed migration at approximately 125 kDa in SDS-PAGE . Bands significantly deviating from this size may represent non-specific binding or FAM120A isoforms/modifications.

• siRNA validation: Compare detection in control samples versus FAM120A siRNA-treated samples. Specific signals should show substantial reduction in knockdown samples, as demonstrated in validation data .

• Expected localization patterns: In IF/ICC applications, FAM120A shows predominantly cytoplasmic localization . Unexpected patterns (such as exclusive nuclear staining) should be interpreted cautiously and validated through additional methods.

• Cross-validation with alternative antibodies: When possible, confirm key findings using antibodies from different suppliers or those recognizing different FAM120A epitopes.

• Peptide competition: Pre-incubation with the immunizing peptide should substantially reduce specific signals while leaving non-specific binding relatively unchanged.

• Tissue expression profiling: Compare your expression data with established FAM120A expression patterns. For example, strong cytoplasmic positivity in glandular cells of human colon is consistent with FAM120A expression .

How should I interpret discrepancies in FAM120A detection across different experimental methods?

When faced with inconsistent results between different detection methods, consider these methodological factors:

• Protein conformation differences:

  • Western blot detects denatured proteins, while IF/IHC detect proteins in more native conformations

  • Epitope accessibility may differ significantly between applications

  • Some antibodies perform better in certain applications due to epitope recognition characteristics

• Sample preparation variables:

  • Fixation methods in IHC/IF can mask epitopes or create artifacts

  • Lysis conditions for Western blot may extract protein fractions differentially

  • Antigen retrieval methods significantly impact IHC results; compare results using both recommended methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Expression threshold differences:

  • Western blot and IHC/IF have different detection sensitivities

  • Low expression levels might be detected by one method but not another

• Resolution considerations:

  • IHC/IF provide spatial information that Western blot lacks

  • Western blot can distinguish proteins by size in ways IHC/IF cannot

• Methodological approach:

  • Start with the application where the antibody shows strongest validation

  • Use that as a reference point to optimize other applications

  • Consider that the recommended dilution ranges vary significantly between applications (1:500-1:2000 for WB vs. 1:20-1:200 for IHC)

How can FAM120A antibodies be adapted for single-cell analysis techniques?

While traditional bulk analysis methods provide valuable insights, adapting FAM120A detection to single-cell techniques offers enhanced resolution of cellular heterogeneity:

• Single-cell immunofluorescence approaches:

  • The validated IF/ICC applications (dilution 1:50-1:500) provide a starting point for single-cell imaging

  • For flow cytometry adaptation, begin with the higher antibody concentrations (closer to 1:50) and optimize fixation/permeabilization conditions specifically for suspension cells

  • For imaging mass cytometry or multiplexed immunofluorescence, test antibody performance in combination with other markers and optimize signal amplification if needed

• Proximity ligation assays (PLA):

  • For investigating FAM120A protein interactions at single-cell resolution

  • Combine FAM120A antibody with antibodies against suspected interaction partners

  • Requires antibodies from different host species or isotypes for standard PLA protocols

• Single-cell Western blot considerations:

  • Higher antibody concentrations (closer to 1:500 than 1:2000) may be needed due to the reduced protein quantity per cell

  • Signal amplification systems may be beneficial for detecting lower-abundance proteins

• Imaging optimization:

  • When designing multiplexed imaging panels, consider the predominantly cytoplasmic localization of FAM120A

  • This localization pattern can help distinguish FAM120A signals from nuclear markers in co-staining experiments

What are the considerations for using FAM120A antibodies in translational research studies?

When extending FAM120A research into translational contexts, researchers should consider these methodological aspects:

• Tissue microarray (TMA) applications:

  • The validated IHC protocols (1:20-1:200 dilution) can be applied to TMA analysis

  • Antigen retrieval is critical; follow the recommended TE buffer pH 9.0 method, with citrate buffer pH 6.0 as an alternative if needed

  • Include control tissues (like human skeletal muscle) on TMA blocks to ensure consistent staining across batches

• Biomarker development considerations:

  • Standardize scoring systems for FAM120A expression in tissue samples

  • Document subcellular localization patterns (predominantly cytoplasmic in most cell types)

  • Consider dual staining with cell-type specific markers to characterize expression in heterogeneous tissues

• Clinical sample handling:

  • Pre-analytical variables (fixation time, processing protocols) may affect FAM120A detection

  • Validation across multiple preservation methods (FFPE, frozen sections) may be necessary

  • Consider the impact of storage time on antigenicity, particularly for archival samples

• Quantitative analysis approaches:

  • Develop standardized image analysis protocols for FAM120A quantification

  • Consider digital pathology approaches for objective assessment of expression levels

  • Document scoring reproducibility between observers for subjective assessment methods

These considerations help ensure that FAM120A antibody-based methods can be reliably translated from basic research into clinical research applications.