fam214a Antibody

What detection methods are validated for FAM214A antibodies?

FAM214A antibodies have been validated for several detection methods in research settings. Current commercially available antibodies are primarily validated for immunohistochemistry (IHC), immunohistochemistry-paraffin (IHC-P), and immunofluorescence techniques . For quantitative detection, ELISA-based methods are validated for measuring FAM214A in serum, plasma, and tissue homogenates with detection ranges typically between 0.781-50 ng/mL and minimum detection limits around 0.781 ng/mL .

When designing experiments, researchers should select the appropriate methodology based on their specific research question:

• For tissue localization studies: IHC or IHC-P (recommended dilution 1:20-1:50)

• For cellular localization: Immunofluorescence

• For quantification in biological fluids: ELISA

What are the optimal sample preparation protocols for Western blot detection of FAM214A?

For optimal Western blot detection of FAM214A, the following protocol has been validated in research settings:

• Lyse cells in RIPA buffer supplemented with 2 mM sodium vanadate and protease inhibitor cocktails

• Separate cell lysates (10-15 μg) by gradient 4-20% SDS-PAGE

• Transfer proteins onto PVDF membranes

• Block membranes with appropriate blocking buffer (e.g., Odyssey Blocking buffer) for 1 hour at room temperature

• Incubate with primary rabbit antibodies against FAM214A (1:1000 dilution) overnight at 4°C

• Wash with TBS containing 0.1% Tween-20 (TBST)

• Incubate with appropriate secondary antibody (e.g., IRDye 680RD goat anti-rabbit IgG at 1:10,000 dilution) for 1 hour at room temperature

• For loading control, use β-actin primary antibody (1:5000 dilution)

• Image using an appropriate imaging system

This protocol has been successfully used to detect FAM214A in human samples, with normalization to β-actin for quantitative analysis.

How does sample type affect FAM214A detection and quantification?

The detection and quantification of FAM214A can vary depending on the biological sample type. For ELISA-based detection:

For all sample types, a sample volume of 50 μL is typically required for ELISA-based detection methods . Researchers should be aware that cross-reactivity between FAM214A and analogues might exist, although commercially available assays report high specificity for FAM214A detection .

How can FAM214A antibodies be used to investigate its role in inflammatory processes?

FAM214A has demonstrated differential expression in inflammatory versus anti-inflammatory macrophages, making it an intriguing target for inflammation research. To investigate this role:

• Design experiments comparing FAM214A levels in different macrophage populations:

  • Use FAM214A antibodies to detect expression levels in M1 (inflammatory) versus M2 (anti-inflammatory) macrophages

  • Compare with appropriate polarization markers to confirm macrophage phenotype

• Methodology for investigating inflammatory regulation:

  • Stimulate macrophages with appropriate polarizing factors (e.g., LPS/IFN-γ for M1, IL-4/IL-13 for M2)

  • Use Western blot or immunofluorescence with FAM214A antibodies to quantify expression changes

  • Consider time-course experiments to track dynamic expression changes

Research has shown that FAM214A levels are elevated in inflammatory M1-differentiated mouse bone marrow macrophages and reduced in anti-inflammatory M2-differentiated macrophages . This differential expression suggests FAM214A may play a role in inflammatory response pathways and could be a target for inflammatory disease studies.

What controls should be included when using FAM214A antibodies in immunohistochemistry experiments?

When designing immunohistochemistry experiments with FAM214A antibodies, include these essential controls:

• Positive controls:

  • Tissues known to express FAM214A (based on research findings)

  • Cell lines with validated FAM214A expression

• Negative controls:

  • Primary antibody omission (to detect non-specific binding of secondary antibody)

  • Isotype control (matched immunoglobulin at the same concentration)

  • Tissues or cells known not to express FAM214A

• Specificity controls:

  • Pre-absorption with immunizing peptide (the antibody's specific immunogen sequence is: TNEGKIRLKPETPRSETCISNDFYSHMPVGETNPLIGSLLQERQDVIARIAQHLEHIDPTASHIPRQSFNMHDSSSVASKVFRSSYEDKNLLKKNKDESSVSISHT)

  • If available, FAM214A knockout or knockdown samples

• Technical controls:

  • Include serial dilutions of primary antibody to determine optimal concentration

  • Use standardized positive tissues across experimental batches

For advanced applications, consider dual-labeling experiments with markers of cellular compartments to determine FAM214A subcellular localization.

How can researchers investigate the functional relationship between FAM214A and purine metabolism?

Research has identified FAM214A as a novel component of the conserved uric acid pathway relevant for metabolic diseases and aging . To investigate this functional relationship:

• Experimental approach using FAM214A antibodies:

  • Perform immunoprecipitation using FAM214A antibodies followed by mass spectrometry to identify interacting proteins in the purine metabolism pathway

  • Use proximity ligation assays with FAM214A antibodies and antibodies against known purine metabolism proteins to confirm in situ interactions

• Functional studies:

  • Manipulate FAM214A expression (knockdown/overexpression) in relevant cell models

  • Measure changes in purine metabolites using metabolomics approaches

  • Correlate FAM214A protein levels (detected with antibodies) with metabolite concentrations

• Metabolic flux analysis:

  • Use isotope-labeled precursors to track purine synthesis in systems with altered FAM214A expression

  • Measure phosphoribosyl pyrophosphate (PRPP), uric acid, and guanosine levels as indicators of purine metabolism changes

Research has shown that modulation of the FAM214A homolog in Drosophila (mda) affects purine metabolism precursors, suggesting a conserved role across species .

What are common challenges in detecting FAM214A in clinical samples and how can they be addressed?

Researchers often encounter several challenges when detecting FAM214A in clinical samples:

• Low abundance issues:

  • Solution: Use signal amplification methods like tyramide signal amplification for IHC

  • Consider more sensitive detection methods like ELISA (with detection limits around 0.781 ng/mL)

• Non-specific binding:

  • Solution: Optimize blocking protocols using different blocking agents

  • Increase wash steps and duration

  • Validate antibody specificity using peptide competition assays

• Sample degradation:

  • Solution: Process samples immediately after collection

  • Use appropriate protease inhibitors in all buffers

  • Store samples at recommended temperatures (-80°C for long-term storage)

• Inconsistent results across samples:

  • Solution: Standardize sample collection and processing procedures

  • Use internal controls for normalization

  • Consider batch effects in analysis

For ELISA-based detection specifically, automated plate washing is recommended to ensure consistent washing steps across all wells, with a recommended soaking time of 1 minute to reduce background signal .

How can researchers optimize Western blot protocols for detecting low levels of FAM214A?

For detecting low levels of FAM214A in biological samples, researchers can implement these optimization strategies:

• Sample enrichment techniques:

  • Increase protein loading (up to 30 μg may be required for low-abundance samples)

  • Consider immunoprecipitation to concentrate FAM214A before Western blotting

• Signal enhancement strategies:

  • Use high-sensitivity chemiluminescent substrates

  • Consider longer exposure times (with appropriate controls for background)

  • Use fluorescent secondary antibodies with infrared imaging systems for better quantification

• Protocol modifications:

  • Extend primary antibody incubation time (overnight at 4°C is standard, but up to 48 hours may improve signal)

  • Use PVDF membranes instead of nitrocellulose for better protein retention

  • Reduce washing stringency slightly (use 0.05% instead of 0.1% Tween-20)

• Antibody optimization:

  • Test different antibody concentrations (ranging from 1:500 to 1:2000)

  • Try different antibody clones if available

  • Consider using a cocktail of antibodies targeting different epitopes

The validated protocol detecting FAM214A normalized to β-actin using infrared imaging systems has proven effective in research settings , but may require further optimization for specific sample types.

What strategies can address cross-reactivity issues when using FAM214A antibodies?

Cross-reactivity can complicate the interpretation of results when using FAM214A antibodies. Researchers can employ these strategies to address such issues:

• Epitope analysis:

  • Review the immunogen sequence used to generate the antibody (for available antibodies, this sequence is: TNEGKIRLKPETPRSETCISNDFYSHMPVGETNPLIGSLLQERQDVIARIAQHLEHIDPTASHIPRQSFNMHDSSSVASKVFRSSYEDKNLLKKNKDESSVSISHT)

  • Use bioinformatics tools to identify potential cross-reactive proteins with similar epitopes

• Validation approaches:

  • Perform peptide competition assays using the specific immunizing peptide

  • Test the antibody on samples with FAM214A knockdown or knockout

  • Use multiple antibodies targeting different epitopes of FAM214A

• Experimental design considerations:

  • Include appropriate negative controls in all experiments

  • Consider using more specific detection methods like targeted mass spectrometry for validation

  • Increase washing stringency in protocols to reduce non-specific binding

• Data analysis strategies:

  • Use appropriate software to analyze band patterns in Western blots

  • Consider molecular weight discrepancies that might indicate cross-reactivity

  • Document all unexpected bands for further investigation

Commercial FAM214A antibodies and ELISA kits report high specificity, though manufacturers acknowledge limitations in complete cross-reactivity testing .

How does FAM214A expression correlate with hyperuricemia and gout pathology?

Research has identified FAM214A as a component of the conserved uric acid pathway with potential implications for hyperuricemia and gout:

• Experimental evidence from model systems:

  • Studies in Drosophila show that the FAM214A homolog (mda) regulates phosphoribosyl pyrophosphate (PRPP), uric acid, and guanosine levels

  • Knockdown of mda limits the formation of uric acid concretions in Drosophila models of hyperuricemia

• Cellular mechanisms:

  • FAM214A influences intracellular uric acid levels in human HepG2 transformed hepatocytes

  • Its expression is differentially regulated in inflammatory versus anti-inflammatory macrophages, suggesting a role in inflammation associated with hyperuricemia

• Research applications:

  • FAM214A antibodies can be used to study protein expression in tissues from gout patients

  • Correlation studies between FAM214A levels (detected via ELISA or Western blot) and serum urate levels may provide insights into disease mechanisms

These findings suggest FAM214A is a potential target to ameliorate negative effects of elevated uric acid burden without the detrimental consequences of targeting upstream genes with pleiotropic effects .

What is the subcellular localization of FAM214A and how can antibodies help determine its functional compartmentalization?

Understanding the subcellular localization of FAM214A is crucial for elucidating its function:

• Immunofluorescence approach:

  • Use FAM214A antibodies in combination with markers for different cellular compartments

  • Perform confocal microscopy to precisely determine co-localization

  • Consider live-cell imaging with fluorescently tagged antibody fragments for dynamic studies

• Biochemical fractionation validation:

  • Perform subcellular fractionation to isolate different cellular compartments

  • Use Western blotting with FAM214A antibodies to determine enrichment in specific fractions

  • Compare with known markers of cellular compartments

• Advanced localization techniques:

  • Consider super-resolution microscopy for precise localization

  • Use electron microscopy with immunogold-labeled FAM214A antibodies for ultrastructural localization

  • Perform proximity ligation assays to identify proteins in close association with FAM214A

Current research has not fully characterized the subcellular localization of FAM214A, making this an important area for investigation to better understand its biological function.

How can FAM214A antibodies be used to investigate its role in aging-related pathologies?

Research has suggested a role for FAM214A in aging-related pathologies, particularly through its involvement in purine metabolism and inflammatory processes:

• Age-related expression changes:

  • Use FAM214A antibodies to compare protein expression in tissues from young versus aged organisms

  • Correlate expression levels with markers of aging or senescence

  • Examine FAM214A levels in models of accelerated aging

• Intervention studies:

  • Assess changes in FAM214A expression following interventions known to influence lifespan

  • Use antibodies to monitor FAM214A levels in longitudinal studies of aging interventions

  • Correlate changes with functional outcomes and biomarkers of aging

• Disease-specific applications:

  • Investigate FAM214A expression in tissues affected by age-related conditions like inflammatory arthropathies

  • Compare FAM214A levels in normal versus diseased tissues using immunohistochemistry

  • Consider multiplex approaches to simultaneously detect FAM214A and markers of cellular senescence

The role of FAM214A in aging-related pathologies is supported by research showing that knockdown of its homolog in Drosophila rescued lifespan reduction in models of hyperuricemia , suggesting a conserved role across species that warrants further investigation.

How can multi-omics approaches incorporating FAM214A antibody-based techniques advance understanding of its biological function?

Integrating antibody-based detection of FAM214A into multi-omics research frameworks can provide comprehensive insights:

• Integrative experimental design:

  • Combine proteomics (including FAM214A immunoprecipitation) with transcriptomics to correlate protein-level changes with gene expression

  • Integrate metabolomics focusing on purine metabolites with FAM214A protein quantification

  • Use antibody-based tissue imaging with spatial transcriptomics for localized correlation studies

• Data integration strategies:

  • Apply computational approaches to correlate FAM214A protein levels with metabolite profiles

  • Use network analysis to place FAM214A in biological pathways

  • Develop predictive models incorporating protein, transcript, and metabolite data

• Advanced applications:

  • Use FAM214A antibodies for ChIP-seq to identify potential transcriptional regulatory roles

  • Apply proximity labeling techniques with FAM214A antibodies to identify the proximal proteome

  • Consider single-cell approaches combining antibody detection with transcriptomics

These integrated approaches can help resolve the complex biological functions of FAM214A beyond its established role in purine metabolism and inflammation.

What are the considerations for developing and validating new monoclonal antibodies against FAM214A for specialized research applications?

Researchers developing new monoclonal antibodies against FAM214A should consider:

• Epitope selection strategies:

  • Target unique, conserved regions of FAM214A to ensure specificity

  • Use bioinformatics to identify surface-exposed epitopes likely to be accessible in experimental conditions

  • Consider targeting functional domains to develop potentially inhibitory antibodies

• Validation requirements:

  • Perform specificity testing against recombinant FAM214A and related family members

  • Validate on FAM214A knockout/knockdown systems and overexpression systems

  • Test across multiple techniques (Western blot, IHC, IP, ELISA) to characterize performance

• Application-specific optimization:

  • For structural studies: validate epitope accessibility in native protein conformations

  • For therapeutic research: test antibody functionality in relevant disease models

  • For live-cell imaging: evaluate cell permeability or develop membrane-permeable formats

• Quality control parameters:

  • Establish lot-to-lot consistency testing protocols

  • Determine stability under various storage conditions

  • Document cross-reactivity profiles comprehensively

Currently available antibodies have been validated for specific applications , but specialized research questions may require custom antibody development.

How can FAM214A antibodies be used in conjunction with genetic manipulation techniques to elucidate disease mechanisms?

Combining antibody-based detection with genetic manipulation provides powerful approaches for mechanistic studies:

• CRISPR/Cas9 gene editing applications:

  • Generate FAM214A knockout or knockin cell lines

  • Use antibodies to validate editing efficiency at the protein level

  • Apply antibodies to characterize phenotypic changes following gene editing

• RNA interference studies:

  • Design siRNA or shRNA targeting FAM214A

  • Use antibodies to confirm knockdown efficiency

  • Apply to functional studies investigating metabolic or inflammatory phenotypes

• Rescue experiment approaches:

  • After FAM214A knockout, introduce wild-type or mutant versions

  • Use antibodies to confirm expression levels of introduced constructs

  • Correlate expression levels with functional readouts

• Disease model applications:

  • Apply genetic manipulation in disease-relevant cell types or animal models

  • Use antibodies to track FAM214A expression changes throughout disease progression

  • Correlate with metabolic parameters like uric acid levels or inflammatory markers

This combined approach has been productive in model systems, as demonstrated by studies showing that reconstitution of wild-type cells in knockout models can rescue disease phenotypes in other family members , suggesting similar approaches could be valuable for FAM214A research.