FAM105A Antibody

What is FAM105A and what is its cellular localization?

FAM105A (family with sequence similarity 105, member A) is a pseudodeubiquitinase belonging to the ovarian tumor protease (OTU) family. It is the only putative pseudodeubiquitinase of this family in humans . Unlike its structural homolog OTULIN (FAM105B), FAM105A lacks catalytic function against ubiquitin linkages and cannot bind ubiquitin .

FAM105A exhibits a distinctive subcellular localization pattern, predominantly found at the endoplasmic reticulum (ER) membrane and nuclear envelope. Immunofluorescence studies have shown that FAM105A localizes to the cytoplasm, nuclear envelope, and ER membrane-like structures, which is distinctly different from OTULIN's localization pattern at or near the plasma membrane and in the cytoplasm . This localization is likely facilitated by a twenty-residue stretch of conserved hydrophobic amino acids in the N-terminal region, forming a putative protein membrane localization motif (PMLM) .

What applications can FAM105A antibodies be used for?

Based on validated technical data, FAM105A antibodies can be used for several experimental applications:

Researchers should note that applications are antibody-specific, with each commercial antibody validated for particular techniques and sample types .

What is the molecular weight of FAM105A and how does this affect detection?

FAM105A has a calculated molecular weight of 42 kDa (356 amino acids), but is typically observed at approximately 35 kDa in Western blot applications . This discrepancy between calculated and observed molecular weight is important for researchers to recognize when interpreting experimental results.

When performing Western blot analysis, researchers should expect to detect FAM105A at approximately 35 kDa rather than at the theoretical 42 kDa position . This difference may be attributed to post-translational modifications, protein folding characteristics, or possibly proteolytic processing of FAM105A in cellular contexts. Positive controls such as COLO 320 cells, human placenta tissue, or K562 cell lysates can help confirm appropriate band identification .

How does FAM105A's structure explain its classification as a pseudodeubiquitinase?

FAM105A possesses an OTU domain structure similar to active deubiquitinases but contains critical structural deficiencies that render it catalytically inactive. X-ray crystallography at 2.1 Å resolution revealed that FAM105A maintains a canonical OTU fold with structural similarity to OTULIN (RMSD of 1.45Å over 247 ordered Cα atoms) .

Despite this similarity, FAM105A exhibits several key structural deficiencies:

• Impaired catalytic infrastructure - The active site lacks properly positioned catalytic residues required for deubiquitinating activity

• Deficient substrate-binding infrastructure - Critical ubiquitin-binding surfaces are compromised

• Inability to bind mono-ubiquitin or linear di-ubiquitin - In contrast to OTULIN which shows high specificity for M1 ubiquitin chains

These structural characteristics classify FAM105A as a pseudoenzyme—maintaining a similar fold to catalytically active counterparts but lacking functional enzymatic activity. Understanding this distinction is crucial for experimental design when studying FAM105A's cellular functions .

What technical challenges are associated with immunological detection of FAM105A?

Several technical challenges should be considered when designing experiments for FAM105A detection:

• Membrane localization complexity: FAM105A's localization to the ER membrane and nuclear envelope requires careful sample preparation to ensure adequate extraction without disrupting the protein's native conformation .

• Cross-reactivity concerns: Given the structural similarity to OTULIN (FAM105B), antibody specificity must be rigorously validated to ensure selective detection of FAM105A .

• Buffer optimization for IHC: For optimal immunohistochemical detection in human placenta tissue, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative .

• Variable expression levels: FAM105A mRNA is expressed at moderate to high levels across tissues including lung, gastrointestinal tract, prostate, seminal gland, and bone marrow, but protein expression levels may vary significantly .

To address these challenges, researchers should perform thorough antibody validation, optimize extraction protocols for membrane proteins, and include appropriate positive controls (COLO 320 cells, human placenta tissue, or K562 cells) in their experimental design .

How can researchers differentiate between FAM105A and FAM105B (OTULIN) in experimental systems?

Distinguishing between these structurally similar but functionally distinct family members requires careful experimental design:

• Subcellular localization analysis: Co-localization studies with organelle markers can help differentiate FAM105A (ER/nuclear envelope) from OTULIN (plasma membrane/cytoplasm). Using KDEL markers for ER and Lamin B1 for nuclear envelope can confirm FAM105A localization .

• Functional assays: OTULIN exhibits specific deubiquitinating activity against M1 ubiquitin chains and plays a role in LUBAC-dependent NFκB signaling, while FAM105A lacks these activities. Functional assays measuring deubiquitinating activity can easily differentiate between the two proteins .

• Antibody selection: Choose antibodies raised against unique epitopes. For example, antibody HPA046638 targets the N-terminal region with the immunogen sequence "MAATRSPTRARERERSGAPAAGSDQVHSWMLATSQALDT" , which differs from OTULIN.

• Molecular weight differentiation: On Western blots, FAM105A is observed at approximately 35 kDa, which may differ from OTULIN, aiding in discrimination between the two proteins .

In co-expression studies, epitope tagging strategies (such as HA-tagging FAM105A and FLAG-tagging OTULIN) combined with immunofluorescence analysis can clearly distinguish their respective localization patterns .

What are the optimal protocols for FAM105A antibody applications in Western blotting?

For optimal Western blot detection of FAM105A, the following protocol elements are recommended:

Sample Preparation:

• Use COLO 320 cells, human placenta tissue, or K562 cell lines as positive controls

• Ensure complete lysis to extract membrane-associated FAM105A

• For K562 cell line lysates, load approximately 35 μg of total protein

Antibody Dilutions:

• For antibody 25668-1-AP: Use 1:200-1:1000 dilution

• For antibody ab112961: Use 1:100-1:500 dilution

• Optimize dilutions for each experimental system

Detection Parameters:

• Expected molecular weight: ~35 kDa (observed) despite 42 kDa calculated weight

• Use appropriate molecular weight markers to accurately identify the FAM105A band

• Secondary antibody selection should match the primary antibody host species (typically rabbit IgG)

Storage Conditions:

• Store antibodies at -20°C for long-term storage

• For antibody 25668-1-AP: Stable for one year after shipment; aliquoting is unnecessary for -20°C storage

• For short-term storage (up to 6 months), some antibodies may be stored at 4°C

The specific product protocols from manufacturers should be consulted for detailed step-by-step procedures to ensure optimal results .

What conditions are critical for successful immunohistochemical detection of FAM105A?

Successful immunohistochemical detection of FAM105A requires attention to several key parameters:

Tissue Selection:

• Human placenta tissue has been validated as a positive control for FAM105A detection

• Other tissues with moderate to high FAM105A mRNA expression (lung, gastrointestinal tract, prostate) may also be suitable

Antigen Retrieval:

• Primary recommendation: TE buffer pH 9.0

• Alternative approach: Citrate buffer pH 6.0

• Adequate antigen retrieval is critical due to FAM105A's membrane association

Antibody Parameters:

• For antibody 25668-1-AP: Use 1:20-1:200 dilution

• For antibody HPA046638: Use 1:50-1:200 dilution

• Incubation times and temperatures should be optimized based on tissue type and fixation conditions

Signal Detection:

• Use appropriate detection systems compatible with rabbit IgG primary antibodies

• Include positive and negative controls to validate staining specificity

• Counterstaining should be optimized to visualize nuclear envelope and ER membrane localization

Researchers should note that titration of the antibody concentration may be necessary for each experimental system to obtain optimal signal-to-noise ratio .

How can researchers validate FAM105A antibody specificity?

Validating antibody specificity is crucial for reliable FAM105A research. Recommended approaches include:

• Positive control testing: Confirm detection in validated positive control samples (COLO 320 cells, human placenta tissue, K562 cells) .

• Molecular weight verification: Ensure detection at the expected ~35 kDa band in Western blot applications .

• Subcellular localization analysis: Verify ER membrane and nuclear envelope staining patterns using co-localization with established markers (KDEL for ER, Lamin B1 for nuclear envelope) .

• Knockout/knockdown controls: Utilize FAM105A gene knockout or siRNA knockdown samples to confirm antibody specificity by demonstrating loss of signal.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to demonstrate blocking of specific binding.

• Multiple antibody validation: Compare staining patterns using antibodies raised against different epitopes of FAM105A, such as those targeting N-terminal (HPA046638) versus internal regions (ab112961) .

For immunofluorescence applications, researchers should consider dual staining with antibodies against OTULIN to demonstrate the distinct localization patterns of these related proteins .

What are the functional implications of FAM105A's pseudoenzyme status?

As a pseudodeubiquitinase, FAM105A lacks catalytic activity but may serve important non-enzymatic functions in cellular processes:

• Scaffold protein functions: Despite lacking enzymatic activity, FAM105A may function as a scaffold, recruiting other proteins to specific subcellular locations such as the ER membrane .

• Regulatory roles: FAM105A may regulate active deubiquitinases by competing for binding partners or substrates, similar to other pseudoenzymes in signaling pathways.

• Membrane organization: Its specific localization to the ER membrane and nuclear envelope suggests potential roles in membrane organization or ER-nuclear communication .

• Potential role in metabolism: Some evidence suggests FAM105A may play a role in insulin secretion, indicating functions in metabolic processes .

When interpreting experimental data, researchers should consider these non-enzymatic functions rather than searching for deubiquitinating activity. The BioID interactome profile of FAM105A reveals enrichment of proteins localized to the ER/outer nuclear membrane, Golgi, and vesicular membranes, suggesting involvement in membrane-related processes .

How does FAM105A differ from other OTU family members in experimental systems?

FAM105A exhibits several key differences from other OTU family members that impact experimental approaches:

• Absence of catalytic activity: Unlike most OTU family deubiquitinases, FAM105A lacks catalytic activity against all ubiquitin linkages and cannot bind ubiquitin . Deubiquitinase activity assays will be negative for FAM105A.

• Distinct subcellular localization: While many OTU deubiquitinases are cytosolic or nuclear, FAM105A specifically localizes to the ER membrane and nuclear envelope .

• No role in LUBAC signaling: Unlike OTULIN (FAM105B), which regulates LUBAC-dependent NFκB signaling, FAM105A plays no discernible role in this pathway . NFκB signaling assays will not show modulation by FAM105A.

• Unique interactome: BioID profiling reveals that FAM105A interacts with a distinct set of proteins compared to other OTU family members, with enrichment for ER/Golgi/vesicular membrane proteins .

These differences necessitate careful experimental design when studying FAM105A, particularly when comparing it to other OTU family members or when interpreting negative results in typical deubiquitinase assays.

What experimental approaches can reveal the biological function of FAM105A?

Given FAM105A's pseudoenzyme status and unique localization, specialized approaches are needed to elucidate its biological functions:

• Proximity labeling techniques: Techniques such as BioID have already revealed significant enrichment of FAM105A-interacting proteins in ER/nuclear, Golgi, and vesicular membranes, providing clues to its functional roles .

• Co-immunoprecipitation coupled with mass spectrometry: This approach can identify stable protein complexes involving FAM105A to elucidate its potential scaffolding functions.

• CRISPR-mediated gene editing: Generation of FAM105A knockout cell lines can reveal phenotypic consequences related to ER membrane function, protein trafficking, or stress responses.

• Domain swapping experiments: Creating chimeric proteins that swap domains between FAM105A and OTULIN can help identify regions responsible for their distinct localizations and functions.

• Tissue-specific expression analysis: Quantitative approaches to measure FAM105A expression across tissues, particularly in lung, gastrointestinal tract, prostate, seminal gland, and bone marrow where mRNA is highly expressed .

• ER stress response assays: Given its ER localization, investigating FAM105A's potential role in ER stress responses could reveal functional significance.

When designing these experiments, researchers should consider the membrane-associated nature of FAM105A and include appropriate controls to account for its pseudoenzyme status and specific subcellular localization .

What storage and handling conditions are critical for FAM105A antibodies?

Proper storage and handling of FAM105A antibodies is essential for maintaining their functionality and specificity:

Researchers should follow manufacturer-specific recommendations for each antibody product, as formulations and optimal handling conditions may vary .

What controls should be included when studying FAM105A?

Comprehensive experimental design for FAM105A studies should include multiple controls:

Positive Sample Controls:

• COLO 320 cells and human placenta tissue for Western blot and IHC

• K562 cell line lysates for Western blot

Negative Controls:

• Secondary antibody-only controls to assess background

• FAM105A knockout or knockdown samples (when available)

• Non-expressing tissues or cell lines

Specificity Controls:

• Peptide competition assays

• Comparison with OTULIN (FAM105B) expression patterns to confirm distinct localization

Subcellular Localization Controls:

• KDEL markers for ER co-localization

• Lamin B1 for nuclear envelope co-localization

• Comparison with OTULIN, which localizes to plasma membrane and cytoplasm

Including these controls will help validate experimental findings and distinguish FAM105A-specific effects from potential artifacts or cross-reactivity issues.

How can researchers optimize immunofluorescence detection of FAM105A's unique subcellular localization?

To optimize visualization of FAM105A's distinctive ER membrane and nuclear envelope localization:

• Fixation methodology:

  • Choose fixation methods that preserve membrane structures (4% paraformaldehyde is often suitable)

  • Avoid harsh permeabilization that may disrupt membrane integrity

• Antibody concentration:

  • For antibody HPA046638: Use 0.25-2 μg/mL

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

• Co-localization markers:

  • Include KDEL markers for ER co-localization

  • Use Lamin B1 for nuclear envelope co-localization

  • Consider dual staining with OTULIN to demonstrate distinct localization patterns

• Imaging parameters:

  • Use confocal microscopy for precise localization

  • Z-stack imaging to fully capture membrane distributions

  • Super-resolution techniques may provide enhanced visualization of membrane associations

• Epitope tag strategies:

  • N-terminal HA-tagging of FAM105A has been successfully used for subcellular localization studies

  • Ensure tags do not interfere with the N-terminal PMLM that may direct membrane localization

When interpreting results, researchers should be mindful that overexpression systems may sometimes alter natural localization patterns, and validation with endogenous protein detection is recommended where possible .