FAM114A2 Antibody

What is FAM114A2 and where is it primarily localized in cells?

FAM114A2 (Family with Sequence Similarity 114 Member A2, also known as C5orf3) is a protein that primarily localizes to the cytoplasm as demonstrated by immunofluorescent staining. Recent research has revealed that FAM114A proteins function as adaptors for the recycling of Golgi enzymes. More specifically, FAM114A2 associates with the cis side of the Golgi stack and interacts with various Golgi-resident membrane proteins . When expressing tagged versions of FAM114A2, such as GFP-tagged forms, the protein accumulates on the Golgi apparatus, confirming its subcellular localization and potential functional role in the secretory pathway .

What is the predicted molecular weight of FAM114A2 in Western blot analysis?

When performing Western blot analysis with FAM114A2 antibodies, researchers should expect to observe a band at approximately 55 kDa. This predicted molecular weight has been confirmed in various human cell and tissue lysates, including RT-4 cells, U-251 MG cells, human plasma, liver, and tonsil samples . It's important to note that post-translational modifications or splice variants may occasionally result in bands of different sizes, but the 55 kDa band represents the canonical form of the protein.

What species reactivity can be expected from commercially available FAM114A2 antibodies?

Available FAM114A2 antibodies demonstrate cross-reactivity across multiple species due to high sequence conservation. Commercially available antibodies show confirmed reactivity with human samples, while many also demonstrate reactivity with mouse, cow, dog, guinea pig, horse, rabbit, bat, chicken, hamster, and monkey samples . BLAST analysis indicates high sequence identity (100%) across numerous species including human, gorilla, gibbon, mouse, hamster, elephant, and many others, suggesting broad cross-species utility of these antibodies . Some species show slightly lower sequence homology, such as opossum and platypus (92%), beetle (85%), and Xenopus (84%) .

What are the validated applications for FAM114A2 antibodies in research protocols?

FAM114A2 antibodies have been validated for multiple experimental applications. Primary applications include Western blotting (WB) at recommended dilutions of approximately 1/250, immunohistochemistry on paraffin-embedded tissues (IHC-P) at dilutions around 1/50, and immunocytochemistry/immunofluorescence (ICC/IF) at concentrations of 1-4 μg/ml . The antibodies perform well in detecting endogenous FAM114A2 in various human cell lines, including A-431 cells, where immunofluorescent staining reveals cytoplasmic localization. Researchers should optimize antibody concentrations for their specific experimental conditions, as cellular fixation methods (such as PFA/Triton X-100 treatment) can affect staining efficiency .

How should pull-down assays be designed to study FAM114A2 interactions?

For pull-down assays investigating FAM114A2 interactions, researchers can follow a methodology similar to that used for circFAM114A2 studies. Begin by designing biotinylated probes specific to FAM114A2 along with appropriate negative control probes . Approximately 1×10^7 cells should be harvested after transfection with the appropriate expression constructs. The biotinylated probe should be incubated with streptavidin magnetic beads at room temperature for approximately 2 hours to generate probe-coated beads. Cell lysates are then incubated with these probe-coated beads overnight at 4°C, followed by washing steps. RNA or protein can be extracted using appropriate methods (such as Trizol for RNA) and analyzed by qRT-PCR or Western blotting . For protein interaction studies, GST-tagged forms of FAM114A2 expressed in bacteria have proven effective as baits in pulldowns to isolate binding partners from cell lysates, with subsequent identification via mass spectrometry .

What protocol should be followed for immunofluorescence detection of FAM114A2?

For immunofluorescence detection of FAM114A2, cells should be cultured in appropriate vessels (e.g., 24-well plates), fixed with 4% paraformaldehyde for 15 minutes at room temperature, and washed thoroughly with PBS (3×3 minutes). Cells should then be permeabilized and blocked using 5% BSA with 0.5% Triton X-100 in PBS for 1 hour at room temperature . Primary antibody against FAM114A2 should be applied at the recommended concentration (1-4 μg/ml) in 5% BSA and incubated overnight at 4°C. After washing with PBST (3×3 minutes), cells should be incubated with an appropriate secondary fluorescent antibody (e.g., 488 nm) in 5% BSA for 1 hour at room temperature in a light-protected environment. Nuclear counterstaining can be performed using DAPI for 10 minutes at room temperature. After final washing steps (PBS, 3×5 minutes), samples can be visualized using confocal microscopy .

How does FAM114A2 interact with Rab2 and what implications does this have for Golgi function?

FAM114A2 has been identified as a binding partner for both Rab2A and Rab2B through GST pulldown experiments followed by mass spectrometry analysis . This interaction is significant because Rab2 GTPases are key regulators of membrane trafficking between the ER and Golgi. Unlike FAM114A1, which pulls down relatively few proteins, FAM114A2 specifically enriches a vast array of Golgi-resident membrane proteins, many of which are presumptive intra-Golgi COPI cargo proteins, such as glycosyltransferases . These findings suggest that FAM114A2 may function as an adaptor protein that helps coordinate the recycling of Golgi enzymes through Rab2-dependent pathways. Researchers investigating Golgi trafficking mechanisms should consider FAM114A2 as a potential key player in maintaining Golgi enzyme homeostasis and proper glycosylation processes .

What is the relationship between circular RNA FAM114A2 (circFAM114A2) and cancer progression?

Research has revealed that circFAM114A2, a circular RNA derived from the FAM114A2 gene, is significantly downregulated in urothelial carcinoma of the bladder (UCB) tissue specimens and cell lines . This downregulation appears to have functional consequences, as circFAM114A2 has been identified as a potential tumor suppressor in bladder cancer. Mechanistically, circFAM114A2 may function by interacting with specific miRNAs, as predicted by bioinformatic analyses using tools such as RNAhybrid, miRanda, and TargetScan . Researchers investigating the role of FAM114A2 in cancer should consider both the protein and its circular RNA form, as they may have distinct but potentially related functions in regulating cell proliferation, migration, and invasion. Statistical analysis of clinical samples has shown a correlation between circFAM114A2 expression profiles and clinicopathological features of patients with UCB .

What genetic approaches can be used to study FAM114A2 function in model organisms?

CRISPR-Cas9 gene editing has been successfully employed to investigate FAM114A function in model organisms such as Drosophila. Complete deletion of the entire FAM114A gene in Drosophila resulted in viable and fertile flies, suggesting that the protein may not be essential for basic developmental processes in this organism . For visualization studies, GFP-tagged forms of FAM114A expressed under UAS control have been generated, allowing tissue-specific expression when combined with appropriate Gal4 drivers. For instance, using a fkh-Gal4 driver to express FAM114A-GFP in larval salivary glands revealed accumulation of the protein on the Golgi apparatus . In mammalian systems, plasmids designed to delete FAM114A1 and FAM114A2 can be generated by annealing complementary oligonucleotide pairs encoding single guide RNAs (gRNAs) with appropriate restriction enzyme-compatible overhangs . These approaches allow for detailed functional analysis of FAM114A2 in various model systems.

What factors should be considered when optimizing Western blot protocols for FAM114A2 detection?

When optimizing Western blot protocols for FAM114A2 detection, several factors require careful consideration. First, antibody dilution is critical - most FAM114A2 antibodies work effectively at dilutions around 1/250 for Western blotting . Second, researchers should be aware of the predicted band size of 55 kDa to correctly identify the protein of interest . Third, sample preparation is crucial - validated lysates include various human cell lines (RT-4, U-251 MG) and tissue extracts (plasma, liver, tonsil) . For challenging samples, increasing protein loading amount or using more sensitive detection systems may improve results. Additionally, blocking conditions and incubation times should be optimized based on specific antibody characteristics and sample types. If non-specific bands appear, more stringent washing steps or alternative blocking agents may be necessary. Finally, researchers should validate their results using positive and negative controls, including lysates from cells with known FAM114A2 expression levels or those where the gene has been knocked down or knocked out.

How can specificity of FAM114A2 antibodies be validated in experimental contexts?

Validating the specificity of FAM114A2 antibodies is essential for ensuring reliable experimental results. A comprehensive validation approach should include multiple complementary methods. First, researchers should perform Western blotting using lysates from cells where FAM114A2 has been knocked down via siRNA or knocked out using CRISPR-Cas9, comparing to control cells . The disappearance of the specific 55 kDa band would confirm antibody specificity. Second, immunoprecipitation followed by mass spectrometry can verify that the antibody is indeed capturing FAM114A2. Third, immunofluorescence staining patterns should be compared between control cells and those with reduced FAM114A2 expression. Additionally, the use of multiple antibodies targeting different epitopes of FAM114A2 that produce consistent results provides further validation. For recombinant expression systems, comparing detection of tagged FAM114A2 with both anti-tag and anti-FAM114A2 antibodies offers another layer of validation . Finally, peptide competition assays, where pre-incubation of the antibody with the immunizing peptide blocks specific signal, can provide definitive evidence of specificity.

What statistical approaches are appropriate for analyzing FAM114A2 expression data in clinical samples?

When analyzing FAM114A2 expression data in clinical samples, several statistical approaches are appropriate depending on the experimental design and data characteristics. For comparing expression levels between two groups (such as tumor versus normal tissue), unpaired two-tailed t-tests are commonly employed when data follow normal distribution . For experiments performed in triplicate and repeated three times, means should be reported with standard error of the mean (SEM). When analyzing the correlation between FAM114A2 expression profiles and clinicopathological features of patients, the chi-square test is an appropriate method . For survival analysis, Kaplan-Meier curves with log-rank tests can evaluate the prognostic significance of FAM114A2 expression levels. Multivariate analyses, such as Cox proportional hazards regression, can determine whether FAM114A2 expression is an independent prognostic factor when considering other clinical variables. Statistical significance is typically defined as p < 0.05 . All these analyses can be performed using software packages such as GraphPad Prism (Version 5 or later) or SPSS 25.0, which are widely accepted in the research community .

How might integration of FAM114A2 research with proteomics advance our understanding of Golgi enzyme recycling?

Integration of FAM114A2 research with proteomics approaches offers promising avenues to advance our understanding of Golgi enzyme recycling. Mass spectrometry analysis has already revealed that FAM114A2 interacts with a vast array of Golgi-resident membrane proteins, many of which are presumptive intra-Golgi COPI cargo proteins, such as glycosyltransferases . Future research should employ quantitative proteomics to characterize changes in the Golgi proteome upon FAM114A2 depletion or overexpression. Proximity labeling techniques such as BioID or APEX could map the spatial organization of FAM114A2 and its interacting partners within the Golgi subcompartments with high resolution. Phosphoproteomics could identify regulatory phosphorylation events on FAM114A2 that might modulate its adaptor function. Additionally, systematic analysis of the interactome differences between FAM114A1 and FAM114A2 could reveal why FAM114A2 specifically enriches Golgi-resident proteins while FAM114A1 pulls down relatively few proteins . These proteomics approaches, combined with functional assays of Golgi enzyme activity and localization, would provide mechanistic insights into how FAM114A2 contributes to maintaining proper Golgi function and protein glycosylation.

What are the implications of circFAM114A2 research for developing novel cancer biomarkers or therapeutics?

The discovery that circFAM114A2 is significantly downregulated in bladder cancer and acts as a potential tumor suppressor opens several promising avenues for biomarker and therapeutic development . As a biomarker, circFAM114A2 expression levels could be evaluated in liquid biopsies (blood, urine) to develop non-invasive diagnostic tests for bladder cancer, potentially improving early detection. The stability of circular RNAs in bodily fluids makes them particularly attractive as biomarkers. For prognostic applications, correlation analyses between circFAM114A2 levels and patient outcomes could identify patient subgroups that might benefit from more aggressive treatment. Therapeutically, understanding the mechanism by which circFAM114A2 suppresses cancer progression—particularly its interaction with specific miRNAs as predicted by bioinformatic analyses—could inform the development of RNA-based therapies . Approaches might include delivery of synthetic circFAM114A2 mimics or development of small molecules that enhance endogenous circFAM114A2 expression or function. Furthermore, identification of the miRNA targets of circFAM114A2 could reveal additional nodes in regulatory networks that might be targeted therapeutically. Future research should investigate whether circFAM114A2 downregulation occurs in other cancer types and determine whether its tumor-suppressive functions are tissue-specific or represent a more general mechanism.

How can genetic models incorporating FAM114A2 knockouts advance our understanding of protein function across different organisms?

Genetic models incorporating FAM114A2 knockouts present valuable opportunities to understand this protein's function across evolutionary diverse organisms. While Drosophila lacking FAM114A remain viable and fertile, suggesting non-essential developmental roles in insects , mammalian models might reveal more critical functions due to potential evolutionary divergence. Future research should develop conditional knockout mouse models to investigate tissue-specific roles of FAM114A2, particularly in secretory-intensive tissues where Golgi function is crucial. Tissue-specific and inducible knockout systems would help distinguish between developmental versus adult homeostatic requirements for the protein. Comparative phenotypic analysis between FAM114A1 and FAM114A2 single and double knockouts would reveal potential functional redundancy between these family members. Additionally, introducing human disease-associated variants into model organisms through knock-in approaches could help determine their functional consequences. Cell-type specific transcriptomic and proteomic profiling of knockout models would identify dysregulated pathways when FAM114A2 is absent. Finally, rescue experiments with wild-type versus mutant forms of FAM114A2 in knockout backgrounds would define critical domains and interaction surfaces required for function . Together, these genetic approaches across different model systems would provide comprehensive insights into the evolutionary conservation and divergence of FAM114A2 function.