FAM234A Antibody

What is FAM234A and why is it important in cellular research?

FAM234A (Family with sequence similarity 234 member A) is a protein with multiple cellular functions that are still being elucidated. It is also known by several synonyms including C16orf9, ITFG3 (integrin alpha FG-GAP repeat containing 3), and gs19 . The protein is of research interest due to its potential roles in cellular regulation and signaling pathways that influence cell proliferation, differentiation, and survival . Studying FAM234A may provide insights into fundamental cellular processes and potentially reveal new therapeutic targets for various diseases.

What types of FAM234A antibodies are currently available for research?

Current research tools include both polyclonal and monoclonal antibodies against FAM234A. Polyclonal options such as rabbit-derived antibodies (e.g., CAB16580) offer broad epitope recognition . Monoclonal antibodies like LS-C787713, ABIN395938, and CF505567 provide more specific target binding . The selection between polyclonal and monoclonal depends on the experimental context, with polyclonals offering higher sensitivity but potentially lower specificity compared to monoclonals.

What applications are FAM234A antibodies validated for?

FAM234A antibodies have been validated primarily for Western blot (WB) applications . Some antibodies also demonstrate utility in immunohistochemistry (IHC) and ELISA techniques . The validated applications vary by manufacturer and specific product, with comprehensive validation data available through resources like Antibodypedia, which lists 153 antibodies from 23 providers with varying degrees of validation .

How should I select the appropriate FAM234A antibody for my specific experimental design?

When selecting a FAM234A antibody, consider these methodological factors:

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, ELISA)

• Epitope recognition: Most commonly used antibodies target the region corresponding to amino acids 70-400 of human FAM234A (NP_114428.1)

• Species reactivity: Confirm reactivity with your experimental species (most FAM234A antibodies are validated for human samples)

• Clonality requirements: Choose between polyclonal (broader epitope recognition) or monoclonal (higher specificity) based on your experimental needs

• Detection system compatibility: Ensure compatibility with your visualization method (fluorescent, chemiluminescent, etc.)

What are the optimal storage and handling protocols to maintain FAM234A antibody integrity?

To preserve antibody functionality:

• Storage temperature: Store at -20°C as recommended by manufacturers

• Aliquoting strategy: Divide into single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody performance

• Buffer conditions: Most FAM234A antibodies are supplied in PBS with stabilizers such as glycerol (50%) and preservatives like sodium azide (0.02%)

• Dilution preparation: Prepare working dilutions immediately before use and maintain at 4°C while handling

• Transportation: When moving between laboratories, transport on ice and minimize time at room temperature

What positive and negative controls are recommended for validating FAM234A antibody specificity?

A rigorous validation approach should include:

• Positive controls:

  • Cell lines known to express FAM234A (expression data available through antibody providers)

  • Recombinant FAM234A protein (such as PROTQ9H0X4, which can serve as a reliable positive control)

  • Tissues with documented FAM234A expression

• Negative controls:

  • Isotype control antibodies (rabbit IgG matched to the FAM234A antibody isotype)

  • Antigen pre-absorption (pre-incubating the antibody with recombinant FAM234A)

  • Knockdown or knockout cell lines where FAM234A expression has been reduced or eliminated

What are the optimal dilution ranges for FAM234A antibodies in various applications?

Application-specific dilution guidelines:

• Western blot: Typical working dilutions range from 1:500 to 1:2000

• ELISA: Follow manufacturer recommendations, generally starting at 1:1000

• Immunohistochemistry: For antibodies validated for IHC, ranges typically start at 1:100

Always perform a dilution series experiment to determine optimal concentration for your specific experimental conditions, as the optimal dilution may vary based on:

• Sample type and preparation method

• Detection system sensitivity

• Incubation time and temperature

• Expression level of FAM234A in your samples

How can I optimize Western blot protocols specifically for FAM234A detection?

For optimal Western blot results with FAM234A antibodies:

• Sample preparation:

  • Use RIPA or similar lysis buffers with protease inhibitors

  • Determine appropriate protein loading (20-50 µg total protein per lane)

• Electrophoresis considerations:

  • Use appropriate percentage gel (8-10% SDS-PAGE) for resolving the expected ~59.5 kDa FAM234A protein

  • Include molecular weight markers spanning 40-75 kDa range

• Transfer parameters:

  • Transfer to PVDF or nitrocellulose membranes

  • Optimize transfer time based on protein size (typically 60-90 minutes at 100V)

• Blocking optimization:

  • Test both BSA and non-fat dry milk blocking solutions (3-5%)

  • Block for 1 hour at room temperature or overnight at 4°C

• Antibody incubation:

  • Primary antibody incubation: Start with 1:1000 dilution overnight at 4°C

  • Secondary antibody: Use anti-rabbit IgG conjugated to HRP at 1:5000-1:10000

• Signal detection:

  • Use enhanced chemiluminescence (ECL) detection reagents

  • Adjust exposure times based on signal intensity

What methodological approaches can address weak or nonspecific signal issues with FAM234A antibodies?

When troubleshooting signal problems:

• For weak signals:

  • Increase antibody concentration (decrease dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal enhancement systems (biotin-streptavidin amplification)

  • Increase protein loading (up to 50-75 µg)

  • Optimize antigen retrieval methods for fixed samples

• For nonspecific signals:

  • Increase blocking stringency (5% BSA or milk)

  • Add 0.1-0.3% Tween-20 to washing and antibody dilution buffers

  • Increase washing duration and frequency

  • Decrease antibody concentration (increase dilution)

  • Pre-absorb antibody with recombinant protein to remove cross-reactive antibodies

How can FAM234A antibodies be utilized in multi-protein complex analysis studies?

For investigating protein-protein interactions involving FAM234A:

• Co-immunoprecipitation (Co-IP) approach:

  • Use FAM234A antibodies conjugated to agarose or magnetic beads

  • Cross-link antibody to beads to prevent heavy/light chain interference

  • Prepare lysates under non-denaturing conditions to preserve protein-protein interactions

  • Analyze precipitates for potential binding partners by mass spectrometry or Western blot

• Proximity ligation assay (PLA) methodology:

  • Combine FAM234A antibody with antibodies against suspected interaction partners

  • Use species-specific secondary antibodies with complementary oligonucleotides

  • Analyze fluorescent signals indicating proteins within 40 nm proximity

• Immunofluorescence colocalization:

  • Perform dual immunofluorescence with FAM234A antibody and antibodies against potential interacting proteins

  • Analyze subcellular distribution patterns and overlap coefficients

What strategies can resolve discrepancies in FAM234A detection between different antibodies?

When facing inconsistent results:

• Epitope mapping analysis:

  • Compare immunogen sequences between antibodies (amino acids 70-400 vs. other regions)

  • Determine if epitope accessibility varies under different experimental conditions

• Validation with orthogonal techniques:

  • Complement antibody detection with mRNA expression analysis

  • Use recombinant expression systems with tagged FAM234A

  • Employ CRISPR-mediated tagging of endogenous FAM234A

• Cross-validation with multiple antibodies:

  • Test multiple antibodies recognizing different epitopes

  • Compare monoclonal and polyclonal antibody results

  • Develop a scoring system based on consensus detection

• Post-translational modification considerations:

  • Evaluate if discrepancies relate to detection of differentially modified forms

  • Use phosphatase or deglycosylation treatments before detection

How can FAM234A antibodies be employed in studying protein dynamics and localization?

For advanced spatiotemporal analysis:

• Live-cell imaging approaches:

  • Combine with cell-permeable fluorescent antibody fragments

  • Use in conjunction with fluorescently tagged FAM234A constructs for validation

• Super-resolution microscopy methods:

  • Employ antibodies with bright, photostable fluorophores

  • Optimize fixation and permeabilization for epitope accessibility

  • Use STORM, PALM, or STED microscopy for nanoscale resolution

• FRAP (Fluorescence Recovery After Photobleaching) analysis:

  • Combine antibody-based detection with photobleaching techniques

  • Measure protein turnover rates in different cellular compartments

• Intracellular tracking:

  • Use pulse-chase experiments with temporal antibody labeling

  • Track FAM234A movement through cellular compartments over time

How should researchers interpret variations in FAM234A molecular weight observed in Western blots?

When analyzing apparent molecular weight discrepancies:

• Expected molecular weight: The predicted molecular weight of FAM234A is approximately 59.5 kDa

• Factors contributing to migration differences:

  • Post-translational modifications (phosphorylation, glycosylation)

  • Protein isoforms resulting from alternative splicing

  • Sample preparation conditions (reducing vs. non-reducing)

  • Gel percentage and buffer system variations

• Verification approaches:

  • Compare with recombinant FAM234A protein standards

  • Perform phosphatase or glycosidase treatments to identify modifications

  • Analyze with mass spectrometry to confirm identity

  • Use multiple antibodies targeting different epitopes

What are the key considerations when interpreting FAM234A localization data from immunofluorescence studies?

For accurate localization analysis:

• Fixation and permeabilization effects:

  • Compare results from different fixation methods (paraformaldehyde, methanol, acetone)

  • Evaluate different permeabilization agents (Triton X-100, saponin, digitonin)

• Antibody validation controls:

  • Include peptide competition controls

  • Compare with GFP-tagged FAM234A localization

  • Use cellular fractionation followed by Western blot to confirm subcellular distribution

• Co-localization markers:

  • Use established organelle markers (ER, Golgi, mitochondria, plasma membrane)

  • Calculate quantitative co-localization metrics (Pearson's coefficient, Manders' overlap)

  • Perform z-stack analysis for three-dimensional distribution assessment

• Physiological state considerations:

  • Evaluate localization under different cell cycle phases

  • Compare stressed vs. unstressed conditions

  • Assess before and after relevant stimuli

What experimental limitations should researchers be aware of when using FAM234A antibodies?

Critical limitations to consider:

• Cross-reactivity potential:

  • Most FAM234A antibodies have been validated only against human samples

  • Potential cross-reactivity with related family members

  • Background signal in certain tissues or cell types

• Conformational epitope accessibility:

  • Epitope masking in protein complexes

  • Differential accessibility under various fixation conditions

  • Limited detection of certain protein states or conformations

• Expression threshold detection:

  • Low sensitivity for detecting low abundance expression

  • Need for signal amplification in tissues with minimal expression

  • Quantification limitations at extremely high or low expression levels

• Technical variability sources:

  • Lot-to-lot variation between antibody preparations

  • Differential performance in various buffer systems

  • Time-dependent degradation affecting long-term studies