TRIM36 Antibody

What is TRIM36 and why is it significant for research?

TRIM36 (Tripartite Motif Containing 36), also known as HAPRIN, RNF98, RBCC728, or ANPH, is a member of the tripartite motif (TRIM) family of RING-containing proteins. It functions as a microtubule-associated E3 ubiquitin ligase that plays crucial roles in cytoskeletal organization and cell cycle progression . TRIM36 is particularly important in developmental biology as it is necessary for dorso-ventral axis formation in Xenopus, and bi-allelic mutations in the TRIM36 gene have been linked to anencephaly, a severe neural tube closure defect in humans . Additionally, TRIM36 has been identified as a potential tumor suppressor in prostate cancer, where it inhibits cell proliferation and migration while promoting apoptosis .

The 728 amino acid TRIM36 protein contains several characteristic domains: a RING-type zinc finger, two B box-type zinc fingers, a coiled-coil domain, a fibronectin type-III domain, and a SPRY domain . These structural features enable TRIM36 to mediate protein-protein and protein-DNA interactions, primarily through its zinc-chelating activity . Understanding the functions and interactions of TRIM36 is valuable for research in cytoskeletal dynamics, cell division, developmental biology, and cancer research.

What types of TRIM36 antibodies are available for research applications?

Several types of TRIM36 antibodies are available for research purposes, varying in host species, clonality, and target epitopes:

• Mouse monoclonal antibodies: These include the SS-03 clone that detects TRIM36 protein of human origin and the 2D11 clone raised against a partial recombinant TRIM36 (amino acids 391-499) .

• Rabbit polyclonal antibodies: Various rabbit polyclonal antibodies targeting different regions of TRIM36 are available, including those recognizing amino acids 70-280, 166-215, 310-339, and the C-terminal region .

• Goat polyclonal antibodies: Such as the Abcam ab4836 that has been validated for use in immunohistochemistry on paraffin-embedded marmoset samples .

These antibodies come in different formats: unconjugated, HRP-conjugated, and FITC-conjugated, providing flexibility for various experimental protocols . When selecting a TRIM36 antibody, researchers should consider the specific application, target species reactivity, and the epitope region being targeted based on their research question.

How is TRIM36 expressed in different tissues, and what implications does this have for antibody selection?

TRIM36 demonstrates a distinctive tissue-specific expression pattern that researchers should consider when designing experiments involving TRIM36 antibodies. The protein is predominantly expressed in prostate, testis, and brain, with weaker expression observed in heart, kidney, and lung . Within cells, TRIM36 localizes to the cytoplasm and specifically to the acrosomal region of germ cells and mature sperm, suggesting a significant role in acrosome reaction and fertilization processes .

This tissue expression profile has important implications for antibody selection. For researchers working with reproductive tissues, antibodies validated specifically for testicular or sperm cell applications would be optimal. Similarly, neuroscience researchers may need antibodies validated for brain tissue detection, while cancer biologists might focus on antibodies tested in prostate tissues. Understanding the native expression levels in your tissue of interest is crucial for determining appropriate antibody concentrations and avoiding false negative results in tissues with naturally lower TRIM36 expression.

What experimental techniques can be performed with available TRIM36 antibodies?

TRIM36 antibodies have been validated for multiple experimental techniques, providing researchers with versatile tools for studying this protein across different research contexts:

• Western Blotting (WB): Most available TRIM36 antibodies are validated for western blotting, allowing for protein expression quantification and molecular weight confirmation . This technique is particularly useful for comparing TRIM36 expression levels across different cell lines or experimental conditions.

• Immunoprecipitation (IP): Some antibodies, such as the SS-03 clone, have been validated for immunoprecipitation, enabling the isolation of TRIM36 and its binding partners for further analysis .

• Immunofluorescence (IF) and Immunocytochemistry (ICC): These techniques allow visualization of the subcellular localization of TRIM36, particularly its association with microtubules and mitotic spindles .

• Immunohistochemistry with paraffin-embedded sections (IHCP): Several antibodies are suitable for tissue section analysis, including the goat polyclonal antibody (Abcam ab4836) that has been used at 1:100 dilution on marmoset samples .

• Enzyme-linked immunosorbent assay (ELISA): Some TRIM36 antibodies are specifically validated for ELISA applications, allowing for quantitative detection of TRIM36 in solution .

When selecting an antibody for a specific application, researchers should review the validation data for that particular technique and consider factors such as the recommended dilution, positive control samples, and any specific protocol modifications required for optimal results.

What are the optimal conditions for using TRIM36 antibodies in Western blotting?

For optimal Western blotting results with TRIM36 antibodies, researchers should consider several technical factors that can influence detection sensitivity and specificity:

First, sample preparation is critical when working with TRIM36. Since it is a microtubule-associated protein, using lysis buffers that preserve cytoskeletal components is recommended. Include protease inhibitors in your lysis buffer to prevent degradation of TRIM36, which has a molecular weight of approximately 76-80 kDa (without post-translational modifications) .

For gel electrophoresis, an 8-10% polyacrylamide gel typically provides good resolution of TRIM36. When transferring to membranes, PVDF membranes often yield better results than nitrocellulose for this protein. Blocking with 5% non-fat milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1-2 hours at room temperature is generally effective.

Most TRIM36 antibodies perform optimally at dilutions between 1:500 and 1:2000 for Western blotting, but specific recommendations vary by product . Primary antibody incubation is typically done overnight at 4°C, followed by appropriate secondary antibody incubation (typically 1:5000-1:10000) for 1-2 hours at room temperature.

When selecting positive controls, consider cell lines or tissues known to express TRIM36, such as testis extracts, prostate cancer cell lines (LNCaP, 22Rv1), or brain tissue lysates . For negative controls, TRIM36 knockdown samples using the validated siRNA approaches mentioned in the literature can be valuable .

How can TRIM36 antibodies be used effectively in immunohistochemistry and immunofluorescence?

For effective immunohistochemistry (IHC) and immunofluorescence (IF) applications with TRIM36 antibodies, researchers should optimize several key parameters:

For IHC on paraffin-embedded tissues, antigen retrieval is crucial. Heat-induced epitope retrieval using citrate buffer (pH 6.0) is commonly effective for TRIM36 detection. Literature indicates that a 1:100 dilution of goat polyclonal TRIM36 antibody has been successfully used for IHC on marmoset tissue samples . For human tissues, especially when studying prostate cancer, multivariate logistic regression analysis has shown that TRIM36 immunoreactivity can be an independent predictor of cancer-specific survival , highlighting the diagnostic potential of properly optimized IHC protocols.

For IF applications, fixation methods can significantly impact results. For cultured cells, 4% paraformaldehyde fixation for 15-20 minutes at room temperature generally preserves TRIM36 structure while maintaining its association with microtubules. Permeabilization with 0.1-0.2% Triton X-100 for 5-10 minutes allows antibody access while preserving cellular structures.

When studying TRIM36's association with microtubules and mitotic spindles, co-staining with microtubule markers (such as α-tubulin) can provide valuable context . Since silencing TRIM36 leads to disrupted microtubules and disorganized mitotic spindles , visualizing these structures through IF can be particularly informative in knockdown studies.

For both IHC and IF, appropriate blocking (5-10% normal serum from the species of the secondary antibody) for 1 hour helps reduce background. Primary antibody incubations are typically performed overnight at 4°C, with dilutions ranging from 1:50 to 1:200 depending on the specific antibody and application .

How are TRIM36 antibodies used in cancer research, particularly prostate cancer studies?

TRIM36 antibodies have emerged as valuable tools in cancer research, particularly in prostate cancer studies where TRIM36 appears to function as a tumor suppressor. Immunohistochemical analysis using TRIM36 antibodies has revealed that TRIM36 expression levels correlate with patient prognosis in prostate cancer, with multivariate logistic regression analysis demonstrating that TRIM36 immunoreactivity is an independent predictor of cancer-specific survival .

In functional studies, researchers have used TRIM36 antibodies to validate both overexpression and knockdown experimental models. Western blotting with TRIM36 antibodies has confirmed that overexpression of TRIM36 suppresses cell proliferation and migration in prostate cancer cell lines including LNCaP, 22Rv1, and DU145 . Conversely, knockdown validation using these antibodies has shown that reducing TRIM36 levels suppresses apoptosis while promoting cell proliferation and migration in LNCaP and 22Rv1 cells .

TRIM36 antibodies have also been useful in microarray analysis validation, confirming that apoptosis-related pathways are significantly upregulated by TRIM36 overexpression . Furthermore, researchers have employed these antibodies in combination with TUNEL assays to demonstrate that apoptosis promoted by docetaxel treatment is alleviated in siTRIM36-treated prostate cancer cells , suggesting potential implications for chemotherapy response.

The continued use of specific and well-validated TRIM36 antibodies is likely to further elucidate the mechanistic role of this protein in cancer progression and potentially identify new therapeutic targets or prognostic markers.

What insights have TRIM36 antibodies provided about developmental biology and neural tube defects?

TRIM36 antibodies have been instrumental in advancing our understanding of developmental biology, particularly regarding neural tube formation and axis development. Research using these antibodies has revealed that TRIM36 plays a crucial role in early developmental processes, with bi-allelic mutations in the TRIM36 gene causing anencephaly, a severe neural tube closure defect in humans .

In Xenopus studies, antibodies against TRIM36 have helped establish its necessity for dorso-ventral axis formation , a fundamental process in vertebrate development. Through immunofluorescence studies, researchers have visualized how TRIM36 associates with microtubules and influences their dynamics during crucial developmental stages.

The mechanistic insights gained from these studies suggest that TRIM36's function as a microtubule-associated E3 ubiquitin ligase is central to its developmental role. TRIM36 appears to coordinate growth speed and stability by acting on the microtubules' plus end, thereby impacting cell cycle progression during embryonic development . Immunofluorescence studies using TRIM36 antibodies have shown that silencing TRIM36 leads to disrupted microtubules and disorganized mitotic spindles , providing visual evidence of its cytoskeletal regulatory function.

Researchers investigating neural tube defects continue to use TRIM36 antibodies to examine expression patterns in neural tissues at various developmental stages, helping to establish temporal and spatial requirements for this protein during neurulation. These studies provide critical insights that may eventually inform genetic counseling and potentially therapeutic approaches for developmental disorders associated with TRIM36 dysfunction.

How do TRIM36 antibodies contribute to understanding the protein's role in microtubule dynamics and cell cycle regulation?

TRIM36 antibodies have been pivotal in elucidating the protein's role in microtubule dynamics and cell cycle regulation through various imaging and biochemical approaches. Immunofluorescence studies utilizing these antibodies have clearly demonstrated that TRIM36 associates with microtubules and plays a functional role in maintaining cytoskeletal integrity .

When researchers silenced TRIM36 in HeLa and LN229 cell lines, immunofluorescence with anti-tubulin and anti-TRIM36 antibodies revealed disrupted microtubules and disorganized mitotic spindles . These visualizations directly connected TRIM36 deficiency to cytoskeletal disorganization. The resulting phenotypes included reduced cell proliferation and increased apoptosis, indicating TRIM36's importance in cell cycle progression .

Interestingly, contradictory observations have emerged when examining the effects of TRIM36 overexpression. Some studies report enhanced cell growth when TRIM36 is overexpressed, while others found a markedly decreased growth rate in NIH3T3 cells following TRIM36 overexpression . TRIM36 antibodies have been essential in validating protein expression levels in these seemingly inconsistent experimental models, suggesting that cell type-specific factors or TRIM36 dosage may determine its effects on proliferation.

Further mechanistic insights have come from co-immunoprecipitation studies using TRIM36 antibodies, which have identified an interaction between TRIM36 and CENP-H, a kinetochore protein crucial for chromosome segregation . This interaction provides a potential molecular mechanism for TRIM36's influence on mitosis and cell cycle regulation.

By enabling these diverse experimental approaches, TRIM36 antibodies continue to expand our understanding of how this E3 ubiquitin ligase coordinates microtubule dynamics and cell division, with implications for both normal physiology and disease states where these processes are dysregulated.

What are common challenges when working with TRIM36 antibodies and how can they be addressed?

Researchers working with TRIM36 antibodies may encounter several common challenges that can be addressed through careful optimization and troubleshooting:

One frequent issue is weak or absent signal in Western blotting or immunostaining. This may occur because TRIM36 has tissue-specific expression, being predominantly expressed in prostate, testis, and brain, with weaker expression in heart, kidney, and lung . If working with tissues or cell lines with naturally low TRIM36 expression, consider using more sensitive detection methods (such as enhanced chemiluminescence for Western blotting or signal amplification systems for immunostaining) or increasing the protein load. Additionally, ensure your antibody recognizes the specific TRIM36 isoform present in your experimental system.

Non-specific binding is another common challenge. This can manifest as multiple bands in Western blotting or high background in immunostaining. To reduce non-specific binding, optimize blocking conditions (try different blocking agents such as BSA, normal serum, or commercial blocking buffers), increase the stringency of washes, and carefully titrate antibody concentrations. For Western blotting, non-specific bands may appear due to protein degradation, so ensure complete protease inhibition during sample preparation.

Cross-reactivity with other TRIM family proteins can also occur due to structural similarities, particularly in the conserved RING finger, B-box, and coiled-coil domains. To address this, select antibodies that target unique regions of TRIM36, such as the SPRY domain or fibronectin type-III domain . Validation through positive and negative controls, including TRIM36 knockdown samples , is essential to confirm specificity.

For immunoprecipitation applications, inefficient pull-down may be encountered. This could be addressed by optimizing lysis conditions to ensure TRIM36 is properly solubilized while maintaining its native conformation. Consider using different detergents or lysis buffers depending on whether TRIM36 is being studied in its microtubule-associated state or as a soluble protein.

How can researchers validate the specificity of TRIM36 antibodies for their specific experimental system?

Validating TRIM36 antibody specificity is crucial for ensuring reliable experimental results. Several complementary approaches should be considered:

Genetic validation through knockdown or knockout systems provides the gold standard for antibody specificity. Researchers studying TRIM36 have successfully used siRNA approaches to knock down TRIM36 expression in prostate cancer cell lines . When combined with Western blotting or immunostaining, this approach can confirm that the signal detected by the antibody decreases proportionally with TRIM36 protein levels. A properly validated siRNA system should show at least 70-80% reduction in TRIM36 protein levels.

Overexpression validation is equally valuable, particularly with tagged TRIM36 constructs. Researchers can transfect cells with epitope-tagged TRIM36 (e.g., FLAG, GFP, or Myc-tagged) and perform dual detection with both anti-TRIM36 antibody and anti-tag antibody. Co-localization of signals in immunofluorescence or corresponding bands in Western blotting would support antibody specificity.

Peptide competition assays can be performed by pre-incubating the TRIM36 antibody with the immunizing peptide (when available from the manufacturer). If the antibody is specific, the peptide should block binding to TRIM36 in your samples, resulting in signal loss.

Multi-antibody validation involves testing multiple antibodies targeting different epitopes of TRIM36. Concordant results from antibodies recognizing distinct regions (e.g., N-terminal vs. C-terminal antibodies) increase confidence in specificity. The search results indicate availability of antibodies targeting different TRIM36 regions including AA 70-280, 166-215, 310-339, 391-499, and C-terminal regions , providing options for this approach.

Finally, mass spectrometry validation of immunoprecipitated proteins can provide definitive confirmation that the antibody is capturing TRIM36 rather than cross-reacting with other proteins.

How can TRIM36 antibodies be integrated with other molecular tools for comprehensive protein function studies?

Integrating TRIM36 antibodies with complementary molecular tools creates powerful experimental paradigms for comprehensively studying this protein's functions:

Combining TRIM36 antibodies with live-cell imaging techniques can reveal dynamic aspects of TRIM36 behavior. For example, researchers investigating TRIM36's role in microtubule organization might use TRIM36 antibodies for fixed-cell immunofluorescence to validate observations made with fluorescently-tagged TRIM36 in live cells. This approach bridges static high-resolution imaging with dynamic temporal information.

For studying TRIM36's E3 ubiquitin ligase activity, antibodies can be used alongside ubiquitination assays. Immunoprecipitation with TRIM36 antibodies followed by Western blotting with anti-ubiquitin antibodies can identify ubiquitinated substrates. Similarly, pull-down of potential substrate proteins followed by probing for TRIM36 can confirm interactions. These approaches have been valuable in understanding how TRIM36's enzymatic activity influences cellular processes through targeted protein degradation.

TRIM36 antibodies can be integrated with transcriptomic approaches, as exemplified by studies showing that microarray analysis revealed apoptosis-related pathway upregulation by TRIM36 overexpression . Validation of key proteins in these pathways using antibody-based methods creates a multi-omics picture of TRIM36 function.

In clinical research contexts, TRIM36 antibodies enable correlative studies between protein expression and patient outcomes. Multivariate logistic regression analysis has shown that TRIM36 immunoreactivity serves as an independent predictor of cancer-specific survival in prostate cancer patients , demonstrating how antibody-based detection methods contribute to translational research.

For mechanistic studies, researchers can combine TRIM36 antibodies with proximity ligation assays to visualize and quantify interactions with binding partners such as CENP-H in situ, providing spatial context to biochemical interaction data.

What emerging applications of TRIM36 antibodies might advance understanding of disease mechanisms?

Several emerging applications of TRIM36 antibodies hold promise for advancing our understanding of disease mechanisms, particularly in cancer and developmental disorders:

In cancer research, TRIM36 antibodies may facilitate the development of diagnostic and prognostic tools. Given that TRIM36 immunoreactivity has already been identified as an independent predictor of cancer-specific survival in prostate cancer , refined immunohistochemical protocols using highly specific TRIM36 antibodies could be incorporated into clinical pathology workflows. Multiplexed immunofluorescence approaches combining TRIM36 antibodies with markers of cell proliferation, apoptosis, and androgen receptor signaling could provide a more comprehensive tumor profile to guide treatment decisions.

For developmental disorders, particularly neural tube defects associated with TRIM36 mutations , antibodies could enable detailed mapping of TRIM36 expression patterns during critical developmental windows. Combined with genetic screening, this could improve both diagnosis and risk assessment. Furthermore, patient-derived organoid models of neurodevelopment could be analyzed with TRIM36 antibodies to visualize how specific mutations affect protein localization and function in a three-dimensional context.

Single-cell protein analysis techniques like mass cytometry (CyTOF) or imaging mass cytometry using metal-conjugated TRIM36 antibodies could reveal cell-to-cell variability in TRIM36 expression within heterogeneous tissues. This approach would be particularly valuable for identifying specific cell populations where TRIM36 expression is most critical during development or cancer progression.

Super-resolution microscopy with TRIM36 antibodies may provide unprecedented insights into the protein's interaction with microtubules at the nanoscale level. This could reveal subtle defects in cytoskeletal organization that contribute to disease phenotypes but are invisible to conventional microscopy.

How might advances in antibody technology improve TRIM36 detection and functional analysis?

Advances in antibody technology are poised to enhance both the sensitivity and specificity of TRIM36 detection, while enabling novel functional analyses:

Recombinant antibody technology is producing increasingly specific monoclonal antibodies with reduced batch-to-batch variability compared to traditional hybridoma-derived antibodies. For TRIM36 research, this could mean more reliable detection across different experimental contexts and improved reproducibility between laboratories. Additionally, antibody engineering could produce anti-TRIM36 antibodies with optimized affinity and reduced background binding.

Nanobodies (single-domain antibodies derived from camelid antibodies) against TRIM36 could offer advantages for certain applications due to their small size (~15 kDa compared to ~150 kDa for conventional antibodies). Their compact dimensions would allow better penetration into tissues and access to sterically hindered epitopes, potentially revealing TRIM36 interactions that are masked from conventional antibodies. Furthermore, their genetic encodability makes them suitable for intracellular expression as "intrabodies" to track or manipulate TRIM36 in living cells.

Bifunctional antibodies that simultaneously target TRIM36 and another protein of interest could enable novel functional studies. For example, a bifunctional antibody recognizing both TRIM36 and a component of the ubiquitin-proteasome system could help identify substrates of TRIM36's E3 ligase activity in situ.

Proximity-dependent labeling methods, such as TurboID or APEX2 fused to anti-TRIM36 antibody fragments, could map the protein's interactome in specific cellular compartments. This approach would be particularly valuable for understanding TRIM36's context-specific functions during different cell cycle phases or developmental stages.