THOC5 Antibody, FITC conjugated

What is THOC5 and what are its primary cellular functions?

THOC5, also known as Fms interacting protein (Fmip), C22orf19, or KIAA0983, is a 683 amino acid protein with a molecular weight of approximately 79 kDa that belongs to the THOC5 family . It functions as a component of the THO subcomplex of the TREX complex, which couples mRNA transcription, processing, and nuclear export . THOC5 specifically associates with spliced mRNA (not unspliced pre-mRNA) and is recruited to mRNA in a splicing- and cap-dependent manner . Its localization is dynamic, as it shuttles between the nucleus and cytoplasm in an M-CSF signaling-dependent manner . Recent research has revealed THOC5's novel role in osteoclast differentiation, indicating its importance in bone remodeling processes .

Why would researchers need a FITC-conjugated THOC5 antibody?

FITC-conjugated THOC5 antibodies provide direct fluorescent detection capabilities, eliminating the need for secondary antibodies in immunofluorescence studies . This is particularly valuable when studying THOC5's dynamic subcellular localization, as demonstrated by findings showing that "THOC5 was mainly detected in the nucleus on day 2 and then diffused into both nucleus and cytoplasm on day 3" after M-CSF stimulation . The FITC conjugation enables direct visualization of this shuttling behavior in live or fixed cells, allowing researchers to track temporal changes in THOC5 distribution during cellular differentiation processes or in response to signaling events .

What are the optimal experimental conditions for using THOC5 antibody in immunofluorescence?

For immunofluorescence applications using THOC5 antibodies, dilution ratios of 1:50-1:500 are recommended based on experimental validation . When working with FITC-conjugated variants, researchers should be particularly cautious about photobleaching and take measures to protect the antibody from light exposure during storage and experimental procedures . For antigen retrieval in tissue samples, TE buffer at pH 9.0 is suggested, although citrate buffer at pH 6.0 may serve as an alternative . Positive immunofluorescence results have been validated in HepG2 cells, making this cell line a good positive control for establishing protocols . It is essential to optimize these conditions for each specific experimental system, as antibody performance may vary across different cell types and sample preparation methods.

How should researchers troubleshoot inconsistent THOC5 staining patterns?

Inconsistent staining patterns with THOC5 antibodies can result from several factors. First, verify antibody specificity using appropriate controls (THOC5 knockdown or knockout samples) . Second, consider that THOC5 localization is dynamic and M-CSF signaling-dependent; treating cells with tyrosine receptor inhibitors like Imatinib prior to M-CSF stimulation can reverse THOC5 nuclear translocation . Third, examine the timing of your experiment, as THOC5 localization changes significantly over the course of differentiation (nucleus on day 2, both nucleus and cytoplasm on day 3, and vesicle-like structures in the cytoplasm by day 5) . Finally, ensure proper fixation and permeabilization protocols that preserve both nuclear and cytoplasmic structures without masking epitopes. For challenging applications, titration of the antibody concentration is recommended to determine optimal signal-to-noise ratios .

How can THOC5 antibodies be used to study nuclear-cytoplasmic shuttling?

THOC5 antibodies, particularly FITC-conjugated variants, provide powerful tools for investigating nuclear-cytoplasmic shuttling mechanisms. Research has demonstrated that THOC5 dynamically shuttles between nucleus and cytoplasm in an M-CSF signaling-dependent manner . To study this phenomenon, researchers can employ time-course experiments with immunofluorescence microscopy using dilutions of 1:50-1:500 . The nuclear accumulation of THOC5 can be disrupted using tyrosine receptor inhibitors like Imatinib, which blocks c-FMS activation . For biochemical validation of this shuttling behavior, researchers should perform nuclear-cytoplasmic fractionation followed by western blotting (recommended dilution 1:3000-1:8000) . This approach has revealed that THOC5 localizes mainly to the nucleus on day 2 after M-CSF stimulation, diffuses between nucleus and cytoplasm on day 3, and appears in vesicle-like cytoplasmic structures by day 5 . Co-immunoprecipitation experiments can further elucidate protein interaction partners that may regulate this shuttling behavior.

How can THOC5 antibodies contribute to research on osteoclastogenesis?

THOC5 antibodies serve as essential tools for investigating the newly discovered role of THOC5 in osteoclast differentiation . To study this process, researchers can use THOC5 antibodies in combination with markers of osteoclast formation to monitor the temporal relationship between THOC5 expression, subcellular localization, and osteoclast differentiation stages . Knockdown experiments using siRNAs against THOC5, followed by immunoblotting and immunofluorescence, have revealed that THOC5 facilitates the nuclear translocation of FMS intracellular domains (FICDs) and is required for RANKL-induced NFATc1 expression during osteoclastogenesis . Co-immunoprecipitation assays using THOC5 antibodies have demonstrated that THOC5 binds to FICD in the nucleus at 48 hours after M-CSF stimulation . For such experiments, researchers should optimally use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . These methodological approaches allow researchers to delineate the mechanistic roles of THOC5 in bone remodeling and potential therapeutic targets for bone disorders.

What are the critical controls for validating THOC5 antibody specificity?

Proper controls are essential for validating THOC5 antibody specificity. Positive controls should include cell lines known to express THOC5, such as HEK-293, HeLa, or NIH/3T3 cells for western blotting applications, and HepG2 cells for immunofluorescence . For negative controls, researchers should implement THOC5 knockdown models using siRNAs, which have been successfully used to diminish THOC5 expression in human osteoclast precursor cells . When performing co-immunoprecipitation experiments, IgG isotype controls are necessary to rule out non-specific binding . Additionally, researchers should validate antibody specificity by confirming the expected molecular weight of 79 kDa in western blot applications . For tissue staining, human breast cancer and lung cancer tissues have shown positive IHC results and can serve as positive controls . Finally, peptide competition assays using the immunizing peptide "derived from human THOC5" can provide definitive evidence of antibody specificity .

What are the optimal storage conditions for preserving FITC-conjugated THOC5 antibody activity?

FITC-conjugated THOC5 antibodies require specific storage conditions to maintain optimal activity. These antibodies should be stored at -20°C and remain stable for approximately one year after shipment . The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For the 20μl size formats, the preparation may contain 0.1% BSA . Importantly, FITC conjugates are light-sensitive, so antibodies should be protected from light during storage and handling to prevent photobleaching . While aliquoting is not strictly necessary for -20°C storage according to manufacturer recommendations, it may still be advisable for antibodies that will be used multiple times to avoid freeze-thaw cycles that could degrade the antibody or affect the FITC fluorophore . When working with the antibody, it should be thawed on ice and kept cold during experimental procedures to preserve reactivity.

What species reactivity do THOC5 antibodies demonstrate?

THOC5 antibodies show variable species reactivity depending on the specific product. The polyclonal THOC5 antibody (14862-1-AP) has been tested and validated for reactivity with human, mouse, and rat samples . In contrast, some THOC5 antibodies, like the FITC-conjugated polyclonal variant, may be specifically designed for human reactivity only . When planning cross-species applications, researchers should carefully review the "Tested Reactivity" and "Cited Reactivity" information for each antibody . For instance, while some antibodies may have been tested in multiple species, publication citations might only validate use in a single species (e.g., mouse) . This information is crucial for experimental design, particularly in comparative studies across model organisms. The conservation of THOC5 protein sequence across species can influence antibody cross-reactivity, so epitope mapping or sequence alignment analysis may be helpful when working with less commonly tested species.

How can THOC5 antibodies be used in multiparameter experiments?

FITC-conjugated THOC5 antibodies are particularly valuable for multiparameter experiments due to their direct fluorescent labeling . When designing such experiments, researchers should consider the spectral characteristics of FITC (excitation ~495 nm, emission ~519 nm) to avoid fluorescence overlap with other fluorophores . For flow cytometry applications, FITC-conjugated THOC5 antibodies can be combined with antibodies against surface markers or other intracellular proteins labeled with spectrally distinct fluorophores such as PE, APC, or Cy5. For co-localization studies using confocal microscopy, THOC5-FITC can be paired with red or far-red fluorescent markers to examine relationships with other cellular components. When studying THOC5's interaction with FICDs, researchers have successfully combined THOC5 detection with c-Fms antibodies in co-immunoprecipitation assays . For such complex experimental designs, careful titration of each antibody is essential to minimize background and optimize signal detection. Additionally, appropriate compensation controls must be included when using multiple fluorophores to correct for spectral overlap.

What roles does THOC5 play in disease processes?

THOC5 has emerging significance in various disease processes, particularly in cancer and bone disorders. Research using THOC5 antibodies has revealed positive immunohistochemical staining in human breast cancer and lung cancer tissues, suggesting altered expression in these malignancies . The protein's fundamental role in mRNA processing and export through the TREX complex implies that dysregulation could have far-reaching consequences for cellular gene expression programs . Additionally, THOC5's newly discovered role in osteoclastogenesis indicates potential involvement in bone diseases characterized by abnormal osteoclast activity, such as osteoporosis or Paget's disease . THOC5 deficiency has been shown to result in "defects in hematopoiesis and alteration of other cell" functions, pointing to possible roles in hematological disorders . The protein also interacts with c-FMS (CSF1R), a tyrosine kinase receptor implicated in various cancers and inflammatory diseases . These connections highlight the importance of THOC5 as a research target and the value of specific antibodies for investigating its roles in pathological conditions.

How can researchers investigate THOC5's interaction with the TREX complex?

THOC5 functions as a component of the THO subcomplex within the larger TREX complex, which couples mRNA transcription, processing, and nuclear export . To investigate these interactions, researchers can employ co-immunoprecipitation assays using THOC5 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) followed by western blotting for other TREX components . Immunofluorescence studies using FITC-conjugated THOC5 antibodies (1:50-1:500 dilution) combined with antibodies against other TREX components can reveal co-localization patterns, particularly at sites of active transcription or mRNA processing . For more dynamic analyses, live cell imaging with fluorescently tagged THOC5 can track its recruitment to newly synthesized mRNAs. RNA immunoprecipitation (RIP) assays using THOC5 antibodies can identify the mRNA species preferentially bound by THOC5-containing complexes. To understand the functional consequences of THOC5 within the TREX complex, knockdown experiments followed by mRNA export assays would reveal which specific mRNA subsets depend on THOC5 for efficient nuclear export. These methodological approaches provide complementary information about THOC5's structural and functional roles within the mRNA processing machinery.