FAM185A Antibody

What is FAM185A and what are its key molecular characteristics?

FAM185A (family with sequence similarity 185, member A) is a human protein encoded by the FAM185A gene (Gene ID: 222234). The protein consists of 393 amino acids with a calculated molecular weight of 42 kDa, although it is often observed at both 42 kDa and 30 kDa in experimental settings . The protein is also known as Protein FAM185A, and its UniProt ID is Q8N0U4 . The gene's GenBank accession number is BC029175 .

While the precise function of FAM185A remains under investigation, its expression pattern can provide insights into potential biological roles. According to human protein atlas data, FAM185A exhibits differential expression across various cell types .

What types of FAM185A antibodies are currently available for research?

Several validated antibodies against FAM185A are available for research applications:

These antibodies have been generated using specific immunogens derived from FAM185A protein sequences. For instance, the 20911-1-AP antibody was generated using a FAM185A fusion protein (Ag15041) , while HPA020470 was generated using a specific sequence fragment: RTLKEWTLQVSPFGRLRARLPCHLAVRPLDPLTYPDGDRVLVAVCGVEGGVRGLDGLQVKYDEDLEEMAIVSDTIHPQASVEV .

What are the optimal conditions for using FAM185A antibodies in Western blot applications?

For Western blot applications using FAM185A antibodies, the following methodology is recommended:

• Antibody dilution: The optimal dilution range for 20911-1-AP is 1:500-1:2000 . For HPA020470, a concentration of 0.04-0.4 μg/mL is recommended .

• Sample preparation: FAM185A has been successfully detected in various human cell lines including HeLa, HEK-293, and MCF-7 cells .

• Expected bands: Researchers should anticipate bands at approximately 42 kDa (calculated molecular weight) and 30 kDa (potentially a splice variant or processed form) .

• Optimization note: As with all antibodies, titration in each specific experimental system is highly recommended to obtain optimal results, as signal strength can vary between cell and tissue types .

• Protocol specificity: Follow the manufacturer-specific protocols provided with each antibody for best results. For instance, Proteintech provides specific WB protocols for their FAM185A antibody (20911-1-AP) .

How should FAM185A antibodies be used for immunohistochemistry (IHC) applications?

For optimal immunohistochemistry applications with FAM185A antibodies:

• Antibody dilution: Use 20911-1-AP at 1:20-1:200 for IHC applications . For HPA020470, the recommended dilution is 1:50-1:200 .

• Antigen retrieval: For 20911-1-AP, antigen retrieval with TE buffer pH 9.0 is suggested. Alternatively, citrate buffer pH 6.0 can be used .

• Positive controls: Human hepatocirrhosis tissue and human spleen tissue have been validated as positive controls for FAM185A detection .

• Signal specificity: Careful optimization of antibody concentration is essential to minimize background staining while maximizing specific signal detection.

• Visualization system: Standard detection systems compatible with rabbit antibodies are suitable for visualization of FAM185A staining.

What are the recommended storage conditions for maintaining FAM185A antibody stability and performance?

To ensure optimal stability and performance of FAM185A antibodies:

How can researchers address potential cross-reactivity concerns with FAM185A antibodies?

Cross-reactivity is a significant concern in antibody-based research. For FAM185A antibodies:

• Validation through multiple approaches: Combine different detection methods (e.g., WB, IHC, and IF) to confirm specificity. Consistency across methods increases confidence in antibody specificity .

• Genetic approaches: Consider using CRISPR/Cas9 knockout or siRNA knockdown of FAM185A as negative controls to validate antibody specificity. This is particularly important given the potential for genetic variation in human samples .

• Recombinant expression validation: Some FAM185A antibodies, like HPA020470, have undergone enhanced validation through recombinant expression systems . This validation approach provides stronger evidence for antibody specificity.

• Multiple antibody comparison: Use antibodies from different sources or those targeting different epitopes of FAM185A to confirm results. Consistent results with different antibodies provide stronger evidence for specific detection .

• Pre-absorption controls: Consider performing pre-absorption controls with recombinant FAM185A protein to demonstrate binding specificity of the antibody.

How might genetic variations in FAM185A affect antibody binding and experimental interpretations?

Genetic variations can significantly impact antibody recognition and experimental interpretations:

• Single nucleotide polymorphisms (SNPs): Genetic variations in the FAM185A gene might alter epitopes recognized by antibodies. This is particularly relevant for polyclonal antibodies that recognize multiple epitopes .

• Splice variants: The observation of both 42 kDa and 30 kDa bands suggests potential splice variants or post-translational modifications of FAM185A . Researchers should consider which isoform is relevant to their specific research question.

• Population differences: Consider the genetic background of your biological samples, as different populations may exhibit different frequencies of genetic variants that could affect antibody binding .

• Epitope knowledge: Understanding the specific epitope(s) recognized by your antibody can help predict potential impacts of genetic variations. For example, the immunogen sequence for HPA020470 is known, allowing researchers to assess whether known polymorphisms occur within this region .

• Controls with variant sequences: In cases where specific variants are suspected, consider using recombinant proteins with the variant sequence as controls to assess potential impacts on antibody binding .

What methodological approaches can be used to optimize immunoprecipitation experiments with FAM185A antibodies?

While standard immunoprecipitation (IP) protocols provide a starting point, optimization for FAM185A requires specific considerations:

• Antibody selection: For IP applications, choose antibodies that have been validated for this purpose or those with high affinity. Polyclonal antibodies often perform well in IP due to their recognition of multiple epitopes .

• Lysate preparation:

  • Use buffer systems that maintain native protein conformation while efficiently extracting FAM185A

  • Consider the subcellular localization of FAM185A when selecting extraction methods

  • Include appropriate protease and phosphatase inhibitors to prevent degradation

• Antibody-bead coupling:

  • Pre-clear lysates to reduce non-specific binding

  • Consider crosslinking the antibody to beads to prevent antibody co-elution

  • Optimize antibody:bead ratios for maximum capture efficiency

• Washing conditions:

  • Balance stringency to minimize background while maintaining specific interactions

  • Consider a gradient of salt concentrations in wash buffers to determine optimal conditions

  • Include controls for non-specific binding using isotype-matched control antibodies

• Elution and detection:

  • Optimize elution conditions based on downstream applications

  • Confirm successful precipitation through Western blotting using a different FAM185A antibody that recognizes a different epitope

How can researchers interpret discrepancies between RNA expression data and protein detection of FAM185A?

Discrepancies between RNA and protein levels are common in biological research and require careful interpretation:

• Post-transcriptional regulation: FAM185A may be subject to microRNA regulation or other post-transcriptional mechanisms that affect the correlation between mRNA and protein levels .

• Protein stability and turnover: Differences in protein half-life compared to mRNA stability can lead to temporal disconnections between transcript and protein abundance .

• Detection sensitivity differences: RNA detection methods (e.g., RNA-seq) often have different sensitivity thresholds compared to protein detection methods (e.g., Western blot, IHC) .

• Tissue and cell-type specificity: RNA expression data from single-cell sequencing indicates differential expression across cell types. Consider whether your protein detection method is analyzing the same cell population as your RNA data .

• Methodological validation:

  • Confirm antibody specificity through multiple approaches

  • Consider quantitative protein methods (e.g., mass spectrometry) to validate antibody-based detection

  • When possible, perform RNA and protein analyses on the same samples to minimize biological variability

What are the considerations for using FAM185A antibodies in multiplexed immunofluorescence assays?

Multiplexed immunofluorescence allows simultaneous detection of multiple proteins, requiring specific optimization for FAM185A:

• Antibody compatibility:

  • Ensure primary antibodies originate from different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies (common for FAM185A), consider sequential staining with thorough blocking between rounds

  • Test for potential cross-reactivity between all antibodies in the panel

• Signal optimization:

  • For HPA020470, the recommended concentration for immunofluorescence is 0.25-2 μg/mL

  • Titrate antibody concentrations to balance specific signal against background

  • Consider tyramide signal amplification for weak signals

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap

  • Include single-stain controls for spectral unmixing if using confocal microscopy with spectral detection

  • Consider the autofluorescence profile of your tissue/cells

• Validation controls:

  • Include both positive and negative controls for each target

  • Consider co-localization patterns with known interaction partners as biological validation

• Image acquisition and analysis:

  • Standardize exposure settings across samples

  • Use appropriate image analysis software for quantification

  • Consider machine learning approaches for complex pattern recognition

How can researchers integrate FAM185A antibody data with omics approaches for comprehensive protein characterization?

Integrating antibody-based detection with omics methodologies provides a more comprehensive understanding of FAM185A biology:

• Proteomics integration:

  • Use immunoprecipitation with FAM185A antibodies followed by mass spectrometry to identify interaction partners

  • Compare protein abundance measurements from antibody-based methods with label-free quantification from proteomics

  • Consider targeted proteomics approaches (MRM/PRM) to quantify FAM185A in complex samples

• Transcriptomics correlation:

  • Compare protein expression patterns detected by FAM185A antibodies with RNA-seq or single-cell RNA-seq data from resources like the Human Protein Atlas

  • Investigate potential discrepancies between RNA and protein levels through mechanistic studies

• Epigenomics insights:

  • Correlate FAM185A expression with epigenetic modifications at the gene locus

  • Consider the impact of chromatin state on expression variability across cell types

• Functional genomics:

  • Combine antibody detection with CRISPR screens to place FAM185A in functional networks

  • Use phospho-specific antibodies (if available) to connect FAM185A to signaling networks

• Systems biology approaches:

  • Integrate multiple data types to build predictive models of FAM185A function

  • Use network analysis to identify potential functional modules involving FAM185A

What approaches can address epitope masking or accessibility issues when working with FAM185A antibodies?

Epitope masking can significantly impact antibody detection and requires methodological solutions:

• Optimization of fixation protocols:

  • Compare different fixatives (formalin, methanol, acetone) for their impact on epitope accessibility

  • Test different fixation durations to balance structural preservation with epitope accessibility

  • For particularly challenging epitopes, consider non-crosslinking fixatives

• Antigen retrieval methodologies:

  • For 20911-1-AP, TE buffer pH 9.0 is recommended for antigen retrieval, with citrate buffer pH 6.0 as an alternative

  • Optimize retrieval conditions (temperature, duration) for maximum epitope exposure

  • Consider enzymatic retrieval methods for heavily crosslinked samples

• Denaturation conditions in Western blotting:

  • Test different detergents and reducing agents in sample preparation

  • Compare heat denaturation temperatures and durations

  • Consider native vs. denaturing conditions based on the epitope characteristics

• Antibody combination strategies:

  • Use multiple antibodies recognizing different epitopes of FAM185A

  • Develop sequential or simultaneous staining protocols to maximize detection

• Alternative approaches:

  • Consider protein tagging strategies (e.g., FLAG, HA) for recombinant expression systems

  • For particularly challenging applications, consider proximity ligation assays to amplify weak signals

How can researchers address inconsistent banding patterns when using FAM185A antibodies in Western blot?

Inconsistent banding patterns are a common challenge that requires systematic troubleshooting:

• Multiple band analysis:

  • The observation of both 42 kDa and 30 kDa bands with FAM185A antibodies is expected and may represent different isoforms or post-translational modifications

  • Unexpected bands should be evaluated for specificity through knockdown/knockout controls

• Sample preparation optimization:

  • Test different lysis buffers to ensure complete protein extraction

  • Include appropriate protease inhibitors to prevent degradation

  • Standardize protein quantification and loading amounts

• Transfer efficiency assessment:

  • Verify transfer efficiency using reversible protein stains

  • Optimize transfer conditions for proteins in the molecular weight range of FAM185A

  • Consider transfer method modifications for proteins that may be difficult to transfer

• Blocking optimization:

  • Compare different blocking agents (BSA, milk, commercial blockers) for optimal signal-to-noise ratio

  • Test blocking duration and temperature effects on specific and non-specific binding

• Positive and negative controls:

  • Include lysates from cells known to express FAM185A (e.g., HeLa, HEK-293, MCF-7)

  • Consider recombinant FAM185A as a positive control

  • Include genetic knockdown/knockout samples as negative controls when possible

What potential artifacts should researchers be aware of when interpreting immunohistochemistry results with FAM185A antibodies?

Immunohistochemistry can be prone to artifacts that require careful consideration during interpretation:

• Edge artifacts and tissue damage:

  • Exclude tissue edges and damaged areas from analysis

  • Ensure proper fixation throughout the tissue sample

  • Consider whole-slide imaging for comprehensive assessment

• Endogenous peroxidase activity:

  • Ensure adequate blocking of endogenous peroxidase activity in tissues

  • Use appropriate controls without primary antibody to assess background

  • Consider alternative detection systems for tissues with high peroxidase activity

• Non-specific binding issues:

  • Optimize blocking conditions (time, temperature, blocking agent)

  • Include isotype control antibodies at the same concentration

  • Consider pre-absorption controls with recombinant FAM185A

• Fixation and processing artifacts:

  • Standardize fixation protocols across samples

  • Be aware of potential antigen loss or modification during processing

  • Consider the impact of decalcification on epitope recognition in bone tissues

• Interpretation guidelines:

  • Use validated positive control tissues (e.g., human hepatocirrhosis tissue, human spleen tissue)

  • Establish scoring criteria before analysis

  • Consider double-blind assessment for objective interpretation