FAM134B Antibody

What is FAM134B and why is it important in cellular research?

FAM134B (Family with Sequence Similarity 134, Member B) is a protein that functions as a selective autophagy receptor for the endoplasmic reticulum, controlling a process known as ER-phagy. It plays crucial roles in cellular homeostasis and various pathological conditions. FAM134B has been identified as a tumor suppressor, with its expression changes associated with different pathological stages in colorectal carcinomas . Additionally, FAM134B has been implicated in limiting viral replication, particularly for Ebola virus in mouse models . Given its significance in disease mechanisms, including cancer progression and viral infections, FAM134B has become an important target for research in cell biology, virology, and oncology.

What types of FAM134B antibodies are commercially available for research?

Several types of FAM134B antibodies are available for research applications, including:

• Polyclonal antibodies: Such as the rabbit polyclonal antibody against the middle region of human FAM134B (ABIN2782025), which reacts with multiple species including human, mouse, rat, cow, dog, guinea pig, horse, rabbit, and yeast .

• Recombinant monoclonal antibodies: For example, the rabbit recombinant monoclonal antibody (84773-1-PBS) that comes in a conjugation-ready format in PBS buffer without BSA or azide .

• Application-specific antibodies: Researchers can select antibodies validated for specific applications such as Western blotting, immunohistochemistry, ELISA, and immunoprecipitation .

Most commercially available FAM134B antibodies are derived from rabbit hosts and target different regions of the FAM134B protein, offering researchers options based on their experimental needs and target species.

What applications are FAM134B antibodies typically used for?

FAM134B antibodies are utilized in numerous research applications:

The application determines which antibody is most suitable. For example, some antibodies like 84773-1-PBS are specifically validated for sandwich ELISA and cytometric bead array applications , while others may be more suitable for Western blot or immunohistochemistry.

How should FAM134B antibodies be stored and handled?

For optimal performance and longevity of FAM134B antibodies:

• Storage temperature: Most FAM134B antibodies should be stored at -20°C for long-term storage, though some formulations, like the 84773-1-PBS recombinant antibody, require storage at -80°C .

• Buffer conditions: Antibodies come in various buffer formulations, including PBS-only formats for conjugation-ready applications or with stabilizers for standard research use.

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles by aliquoting the antibody upon first thaw.

• Working dilutions: Prepare fresh working dilutions on the day of the experiment using appropriate diluents compatible with your application.

• Handling: Always handle antibodies using clean pipette tips and sterile tubes to prevent contamination.

Proper storage and handling of FAM134B antibodies are critical for maintaining their specificity and sensitivity in experimental applications, especially for quantitative assays like ELISA where antibody performance directly impacts detection limits.

How does FAM134B function in ER-phagy, and how can antibodies help investigate this mechanism?

FAM134B regulates ER-phagy through a complex mechanism involving ubiquitination and clustering. The protein contains a reticulon homology domain (RHD) where multiple lysine residues can be ubiquitinated . This ubiquitination is critical for FAM134B's function in ER fragmentation and subsequent autophagic degradation.

When investigating this mechanism, researchers can employ FAM134B antibodies in several sophisticated approaches:

• Co-immunoprecipitation experiments: Using FAM134B antibodies for immunoprecipitation (IP) allows the isolation of FAM134B protein complexes to study its interactions with other proteins involved in the ER-phagy pathway, such as LC3B and ubiquitination machinery components .

• Proximity ligation assays: Combining FAM134B antibodies with antibodies against putative interaction partners (like LC3B) to visualize protein-protein interactions in situ.

• Super-resolution microscopy: Using fluorescently-labeled FAM134B antibodies to track the clustering and dynamics of FAM134B during ER-phagy induction.

Research has shown that FAM134B clusters colocalize with LC3B and ubiquitin in ER fragments, suggesting that ubiquitinated FAM134B clusters interact with autophagosomes . The AMFR E3 ubiquitin ligase has been identified as a key regulator of FAM134B ubiquitination and turnover, with AMFR depletion causing increased FAM134B levels and reduced ER-phagy flux .

What are the considerations for using FAM134B antibodies in viral infection studies?

FAM134B plays a significant role in viral replication, particularly for Ebola virus (EBOV). Studies using FAM134B knockout mouse embryonic fibroblasts (MEFs) have demonstrated that FAM134B-dependent ER-phagy functions as an antiviral mechanism, limiting EBOV replication . When using FAM134B antibodies in viral studies, researchers should consider:

• Cell type selection: Different cell types may express varying levels of FAM134B. Mouse embryonic fibroblasts have been successfully used to study FAM134B's role in EBOV replication .

• Viral strain considerations: Studies have shown that both historic (Mayinga) and contemporary (Makona GCO7) strains of EBOV are affected by FAM134B expression levels, but potentially to different degrees .

• Antibody validation in infection models: Ensure that the selected antibody detects FAM134B efficiently in the context of viral infection, as viral proteins might interfere with epitope accessibility.

• Multiplicity of infection (MOI): In EBOV studies, researchers have used MOIs of 0.01 and 1 to examine FAM134B's impact on viral replication .

• Analysis timepoints: When studying viral replication kinetics, multiple timepoints (e.g., 1, 3, 5, and 7 days post-infection) should be examined .

In FAM134B knockout MEFs, researchers observed 1-2 log₁₀ higher production of infectious EBOV compared to wild-type cells, with increased viral protein production (GP and VP40) and greater nucleocapsid lattice accumulation . This suggests that the FAM134B-dependent ER-phagy pathway may be a potential target for antiviral therapeutic development.

How can I optimize immunodetection of FAM134B in different tissues and cell types?

Optimizing immunodetection of FAM134B requires consideration of tissue/cell-specific factors and appropriate experimental conditions:

• Tissue-specific considerations:

  • Brain tissue: Successfully detected with FAM134B antibody 21537-1-AP in mouse brain, often requiring TE buffer pH 9.0 for antigen retrieval

  • Heart tissue: Similar protocols to brain tissue

  • Cancer cells: May show variable expression; FAM134B has been detected in colorectal cancer cells

• Western blot optimization:

  • Dilution: Optimize antibody concentration (typically 1:2000-1:16000)

  • Protein loading: 20-50 μg total protein is often sufficient

  • Positive controls: C2C12 cells, HEK-293 cells, and brain tissues have shown positive FAM134B expression

  • Detection system: Choose based on expression level; chemiluminescence for moderate-high expression, fluorescent detection for quantitative analysis

• Immunohistochemistry optimization:

  • Fixation: Formalin-fixed, paraffin-embedded sections typically work well

  • Antigen retrieval: Critical for optimal staining; TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be an alternative

  • Blocking: Thorough blocking is essential to reduce background

  • Antibody dilution: Start with 1:250 and optimize based on signal strength

• Controls:

  • Positive tissue controls (brain, heart)

  • Negative controls (antibody omission or isotype control)

  • FAM134B knockout tissues/cells as definitive negative controls, when available

The sensitivity of detection varies by application; electrochemical methods have achieved detection limits as low as 10 pg/μL in research settings , while standard immunoassays typically have detection limits in the ng/mL range.

What are the advantages and limitations of using different detection methods for FAM134B?

Various detection methods offer different advantages and limitations for FAM134B research:

Researchers have recently developed an electrochemical approach for FAM134B detection that offers significant advantages over conventional methods. This approach uses differential pulse voltammetry with a [Fe(CN)₆]³⁻/⁴⁻ redox system and utilizes FAM134B antibodies attached to modified screen-printed carbon electrodes . This method demonstrated excellent sensitivity and specificity in analyzing FAM134B in colon cancer cell extracts and serum samples, with good inter-assay reproducibility (%RSD <8.64) .

When selecting a detection method, researchers should consider the specific research question, required sensitivity, sample type, and available resources.

How do I troubleshoot non-specific binding or weak signals when using FAM134B antibodies?

When facing challenges with FAM134B antibody performance, consider these troubleshooting approaches:

• Non-specific binding issues:

  • Increase blocking duration and concentration (5% BSA or 5% non-fat milk)

  • Optimize primary antibody dilution (start with manufacturer's recommendation and adjust as needed)

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Include additional wash steps with higher stringency buffers

  • Consider using highly purified antibodies that have been validated for your specific application

  • Pre-absorb antibodies with relevant control proteins or tissue lysates

• Weak signal problems:

  • Verify FAM134B expression in your sample (use positive controls like C2C12 cells, HEK-293 cells, or brain tissue)

  • Increase protein loading amount (Western blot) or tissue section thickness (IHC)

  • Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (e.g., biotin-streptavidin, tyramide signal amplification)

  • For Western blot: increase exposure time or use more sensitive detection reagents

• Validation strategies:

  • Use multiple antibodies targeting different epitopes of FAM134B

  • Include FAM134B knockout/knockdown controls

  • Perform peptide competition assays to confirm specificity

  • Verify results using alternative detection methods

• Application-specific considerations:

  • For Western blot: ensure complete protein transfer and appropriate membrane type

  • For IHC: test multiple fixation methods and ensure proper deparaffinization

  • For IP: optimize lysis conditions to preserve protein-protein interactions

  • For ELISA: verify compatibility of capture and detection antibody pairs

Antibody dilution optimization is particularly important, as recommended dilutions can vary widely (e.g., 1:2000-1:16000 for Western blot, 1:50-1:500 for IHC) , and must be determined empirically for each experimental system.

How should I design experiments to study FAM134B's role in ER-phagy?

Designing robust experiments to investigate FAM134B's role in ER-phagy requires a multifaceted approach:

• Genetic manipulation strategies:

  • FAM134B knockout models: Use FAM134B-/- MEFs or CRISPR-Cas9-edited cell lines

  • Knockdown approaches: siRNA targeting FAM134B (verify knockdown efficiency by Western blot)

  • Overexpression systems: Wild-type FAM134B and functional mutants (e.g., ΔLIR, ubiquitination site mutants)

• ER-phagy induction methods:

  • Pharmacological induction: Torin 1 treatment has been shown to induce FAM134B-mediated ER-phagy

  • Stress conditions: ER stress inducers like tunicamycin or thapsigargin

  • Starvation conditions: Serum deprivation or amino acid starvation

• Readout measurements:

  • ER morphology changes: Fluorescent markers for ER (e.g., ER-Tracker, GFP-KDEL)

  • Protein turnover: Cycloheximide chase assays to measure FAM134B degradation rates

  • Colocalization studies: Immunofluorescence for FAM134B, LC3B, and ubiquitin

  • Biochemical fractionation: Isolate ER fractions and quantify FAM134B levels

• Protein-protein interaction studies:

  • Co-immunoprecipitation: Use anti-FAM134B antibodies to pull down associated proteins

  • Proximity labeling approaches: BioID or APEX2 fused to FAM134B

  • BiFC (Bimolecular Fluorescence Complementation): V1-FAM134B and V2-FAM134B constructs have been used to study clustering

• Ubiquitination analysis:

  • Detect FAM134B ubiquitination using anti-ubiquitin antibodies after FAM134B immunoprecipitation

  • Mass spectrometry to identify ubiquitination sites (seven ubiquitinated lysine residues have been identified in the FAM134B RHD)

  • Study E3 ligases: AMFR has been identified as regulating FAM134B ubiquitination

Studies have shown that FAM134B clustering and ubiquitination are critical for ER-phagy. When planning experiments, it's important to include both gain-of-function and loss-of-function approaches, as well as appropriate controls, to thoroughly characterize FAM134B's role in this process.

What are the recommended protocols for using FAM134B antibodies in Western blotting?

A detailed Western blotting protocol for optimal FAM134B detection includes:

• Sample preparation:

  • Lyse cells/tissues in RIPA buffer containing protease inhibitors

  • For tissues: homogenize thoroughly; brain tissue is a reliable positive control

  • Quantify protein concentration (BCA or Bradford assay)

  • Mix 20-50 μg protein with Laemmli buffer containing a reducing agent

  • Heat at 95°C for 5 minutes

• SDS-PAGE separation:

  • Use 10-12% polyacrylamide gels (FAM134B is approximately 55 kDa)

  • Include molecular weight markers

  • Run at 100-120V until appropriate separation

• Transfer:

  • Use PVDF membrane (preferred over nitrocellulose for FAM134B)

  • Transfer at 100V for 60-90 minutes or 30V overnight at 4°C

  • Verify transfer with Ponceau S staining

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or 5% BSA in TBST for 1 hour at room temperature

  • Incubate with primary anti-FAM134B antibody (1:2000-1:16000 dilution) overnight at 4°C

  • Wash 3-5 times with TBST, 5-10 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (typically 1:1000-1:5000) for 1 hour at room temperature

  • Wash 3-5 times with TBST, 5-10 minutes each

• Detection:

  • Apply ECL substrate and image using chemiluminescence detection system

  • For quantification, use β-actin (1:10,000) or vinculin (1:1000) as loading controls

• Controls and validation:

  • Positive controls: C2C12 cells, HEK-293 cells, rat brain tissue, mouse brain, mouse cerebellum

  • Negative controls: FAM134B knockout or knockdown samples

  • Expected band size: ~55 kDa for full-length FAM134B

This protocol has been validated for detecting endogenous FAM134B in various cell types and tissues, and should provide reliable results when following the recommended antibody dilutions and controls.

How do I set up a sandwich ELISA for quantitative detection of FAM134B?

Setting up a robust sandwich ELISA for FAM134B quantification requires careful optimization:

• Materials and reagents:

  • Matched antibody pair: Use validated pairs like 84773-1-PBS (capture) and 84773-2-PBS (detection)

  • Standard protein: Recombinant FAM134B protein for standard curve

  • ELISA plates: High-binding 96-well plates

  • Detection system: HRP-conjugated secondary antibody or biotin-streptavidin system

  • Substrate: TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric detection

• Protocol steps:

  • Coating: Dilute capture antibody (e.g., 84773-1-PBS) in coating buffer (typically 1-10 μg/mL) and incubate in plate wells overnight at 4°C

  • Washing: Wash 3-5 times with washing buffer (PBS with 0.05% Tween-20)

  • Blocking: Add blocking buffer (PBS with 1-5% BSA) for 1-2 hours at room temperature

  • Sample addition: Add diluted samples and standards, incubate 1-2 hours at room temperature

  • Washing: Wash 3-5 times

  • Detection antibody: Add diluted detection antibody (e.g., 84773-2-PBS), incubate 1-2 hours

  • Washing: Wash 3-5 times

  • Secondary antibody: Add enzyme-conjugated secondary antibody, incubate 1 hour

  • Washing: Wash 3-5 times

  • Substrate addition: Add TMB substrate, monitor color development

  • Stop reaction: Add stop solution (e.g., 2N H₂SO₄)

  • Measurement: Read absorbance at 450nm with 620nm reference

• Optimization considerations:

  • Antibody concentrations: Titrate both capture and detection antibodies

  • Sample dilutions: Test multiple dilutions to ensure measurements fall within the linear range

  • Incubation times and temperatures: Optimize for maximum sensitivity

  • Blocking conditions: Test different blocking agents to minimize background

• Validation:

  • Standard curve: Ensure R² > 0.98 with a sigmoid curve covering at least 3 logs

  • Spike-and-recovery: Add known amounts of recombinant FAM134B to samples

  • Precision: Calculate intra-assay (%CV <10%) and inter-assay variability (%CV <15%)

  • Specificity: Test related proteins for cross-reactivity

An optimized sandwich ELISA can achieve detection limits in the low pg/μL range, similar to the electrochemical methods that have demonstrated detection of FAM134B protein at concentrations down to 10 pg/μL .

How is FAM134B being studied in cancer research, and what role do antibodies play?

FAM134B has emerged as an important focus in cancer research, particularly in colorectal carcinomas:

• Expression analysis in cancer:

  • FAM134B has been identified as a tumor suppressor in colorectal cancer

  • Its expression changes correlate with different pathological stages of colorectal carcinomas

  • FAM134B expression has been associated with poor survival in colorectal cancer patients

• Antibody applications in cancer research:

  • Tissue microarray analysis: FAM134B antibodies enable high-throughput screening of tumor samples to correlate expression with clinical outcomes

  • Precision medicine approaches: Detecting FAM134B levels in patient samples may help in stratification and personalized treatment decisions

  • Diagnostic development: Novel detection methods using FAM134B antibodies, such as electrochemical approaches, show promise for clinical applications

• Current methodologies:

  • Conventional methods: ELISA, immunostaining, and Western blot are traditionally used for FAM134B detection in cancer samples

  • Emerging approaches: Electrochemical detection methods offer advantages of being rapid, sensitive, and specific for FAM134B protein in both biological samples (colon cancer cell extracts) and clinical samples (serum)

• Technical achievements:

  • Detection sensitivity: Electrochemical methods have achieved detection limits down to 10 pg/μL with good inter-assay reproducibility (%RSD <8.64)

  • Time efficiency: Newer methods reduce the turnaround time compared to conventional immunoassays

  • Clinical sample compatibility: Methods have been validated with serum samples, enabling potential clinical applications

These advances in FAM134B detection methodologies may lead to low-cost alternatives to conventional immunological assays for point-of-care applications in cancer diagnostics and monitoring .

What are the challenges in studying FAM134B in neurological disorders?

FAM134B has been implicated in neurological disorders, presenting several technical and experimental challenges:

• Technical challenges:

  • Tissue accessibility: Brain tissue is difficult to access in living patients

  • Protein expression levels: FAM134B may be expressed at variable levels in different brain regions

  • Blood-brain barrier: Limits the utility of peripheral biomarkers for central nervous system processes

  • Post-mortem changes: Can affect protein stability and detection in autopsy specimens

• Experimental considerations:

  • Animal models: Must carefully validate relevance to human disease

  • Primary neuronal cultures: Require special handling and have limited lifespan

  • Tissue preservation: Critical for immunohistochemical detection in brain sections

  • Cellular heterogeneity: Brain contains multiple cell types with potentially different FAM134B functions

• Methodological approaches:

  • Optimized immunohistochemistry: FAM134B antibodies have been successfully used in mouse brain tissue with TE buffer pH 9.0 for antigen retrieval

  • Cell-type specific analysis: Combining FAM134B antibodies with neuronal, glial, or vascular markers

  • Subcellular localization: High-resolution imaging to determine precise localization within neuronal compartments

  • Functional assays: Correlating FAM134B levels with neuronal ER morphology and function

• Disease-specific considerations:

  • Vascular dementia: FAM134B has been linked to this condition, requiring vascular co-localization studies

  • Neuronal disorders: May involve FAM134B's role in ER homeostasis and quality control

  • Progressive conditions: Need for longitudinal studies to track FAM134B changes over disease course

When studying FAM134B in neurological contexts, researchers should take advantage of established positive controls such as mouse brain tissue and mouse cerebellum , and consider using multiple detection methods to validate findings.

How can I use FAM134B antibodies in viral infection research beyond Ebola?

While FAM134B has been well-studied in Ebola virus infections, its role in other viral infections represents an important research frontier:

• Experimental approaches for studying FAM134B in viral contexts:

  • Knockout/knockdown systems: Generate FAM134B-deficient cell lines to study various viral replications

  • Time-course studies: Monitor FAM134B levels during different stages of viral infection

  • Localization analysis: Track FAM134B redistribution during viral infection using immunofluorescence

  • Viral factories: Examine co-localization of FAM134B with viral replication complexes

• Potential viral systems to investigate:

  • Other hemorrhagic fever viruses: Marburg, Lassa, etc.

  • RNA viruses that utilize ER for replication: Flaviviruses (Dengue, Zika), Coronaviruses

  • DNA viruses with ER interactions: Herpesviruses, Poxviruses

  • Hepatitis viruses: HCV relies heavily on ER membranes

• Methodological considerations:

  • Biosafety requirements: Adjust protocols based on the biosafety level of the virus

  • Fixation methods: Optimize for simultaneous detection of viral antigens and FAM134B

  • Infection parameters: Test different MOIs and timepoints as established for EBOV (0.01 and 1 MOI, with 1, 3, 5, and 7 days post-infection assessment)

  • Controls: Include both infected and uninfected cells expressing or lacking FAM134B

• Mechanistic investigations:

  • ER stress response: Measure ER stress markers alongside FAM134B during infection

  • Autophagy flux: Quantify autophagy markers in relation to FAM134B levels

  • Viral protein interactions: Perform co-immunoprecipitation with FAM134B antibodies to identify viral proteins that may interact with FAM134B

  • Competitive dynamics: Assess whether viral proteins compete with or modify FAM134B's interactions with the autophagy machinery

When extending FAM134B research to other viral systems, researchers should build upon the established protocols from EBOV studies, where FAM134B knockout resulted in significantly higher viral replication, suggesting a broader antiviral role for the FAM134B-dependent ER-phagy pathway .