TRIM33 Antibody, Biotin conjugated

What is TRIM33 and what are its primary biological functions?

TRIM33 (Tripartite motif-containing 33), also known as KIAA1113, RFG7, or TIF1G, is an E3 ubiquitin ligase and member of the TRIM family. It contains a characteristic conserved RBCC domain and plays crucial roles in multiple biological processes. TRIM33 functions primarily as a regulatory protein that promotes SMAD4 ubiquitination, nuclear exclusion, and degradation via the ubiquitin proteasome pathway. It acts as a transcriptional repressor that inhibits the TGF-beta/BMP signaling cascade and contributes to the control of cell proliferation. Additionally, TRIM33 participates in intracellular signaling, cell development, apoptosis, innate immune response, autophagy, and oncogenesis by forming complexes with various proteins .

What are the structural characteristics of TRIM33 protein?

TRIM33 belongs to the TRIM/RBCC family, containing the tripartite motif domain that mediates protein-protein interactions. The calculated molecular weight of TRIM33 is 122 kDa, though it is typically observed at 140-150 kDa in experimental conditions, likely due to post-translational modifications. The RBCC domain of TRIM33 is particularly important for its function, as demonstrated by studies showing that this domain mediates interaction with other proteins such as PML. TRIM33's gene is located at GenBank accession number NM_015906, with NCBI gene ID 51592 and UniProt ID Q9UPN9 .

How does biotin conjugation affect TRIM33 antibody applications?

Biotin conjugation to TRIM33 antibodies provides advantages in detection sensitivity and versatility across experimental platforms. The biotin-streptavidin system offers signal amplification capabilities that enhance detection sensitivity in applications like ELISA. While unconjugated TRIM33 antibodies are suitable for WB, IP, IF, IHC, and ELISA applications, the biotin-conjugated version (such as CSB-PA891796LD01HU) is particularly optimized for ELISA applications. The biotin conjugation maintains the antibody's specificity for human TRIM33 while adding the advantage of compatibility with streptavidin-based detection systems that can improve signal-to-noise ratios in sensitive assays .

What are the optimal storage conditions for maintaining TRIM33 antibody activity?

For optimal preservation of TRIM33 antibody activity, the biotin-conjugated antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can lead to antibody degradation and reduced activity. The antibody is supplied in a storage buffer containing 0.03% Proclin 300 as a preservative and 50% glycerol in 0.01M PBS at pH 7.4, which helps maintain stability during storage. For frequently used antibodies, it is recommended to prepare small aliquots to minimize freeze-thaw cycles. The antibody should remain stable for at least one year when stored properly at -20°C .

What dilution ranges are recommended for different applications of TRIM33 antibodies?

Though specific dilution recommendations for the biotin-conjugated TRIM33 antibody (CSB-PA891796LD01HU) focus on ELISA applications, data from related TRIM33 antibodies provides guidance for multiple applications. For Western Blot (WB) applications, a dilution range of 1:500-1:1000 is typically recommended. For Immunohistochemistry (IHC), dilutions between 1:50-1:500 are suggested, with antigen retrieval preferably performed using TE buffer at pH 9.0 (alternatively, citrate buffer at pH 6.0 may be used). It is essential to note that optimal dilutions are sample-dependent and should be determined experimentally for each specific system to obtain optimal results .

How should antigen retrieval be performed for optimal TRIM33 detection in tissue samples?

For optimal TRIM33 detection in tissue samples using immunohistochemistry, antigen retrieval methods significantly impact staining quality. The recommended protocol involves using TE buffer at pH 9.0, which has been validated with human lung cancer tissue samples. As an alternative, citrate buffer at pH 6.0 can also be used, though potentially with different efficacy. The antigen retrieval step is crucial for exposing epitopes that may be masked during fixation processes. Temperature and duration of antigen retrieval should be optimized based on tissue type and fixation method, with typical protocols involving heating at 95-100°C for 15-20 minutes followed by cooling to room temperature .

How does TRIM33 interact with the TGF-β signaling pathway in stem cell differentiation?

TRIM33 functions as a key regulator in the TGF-β signaling pathway, particularly during stem cell differentiation to middle endoderm. Research indicates that TRIM33 monoubiquitinates SMAD4, hampering its ability to form stable complexes with activated SMAD2/3, thereby inhibiting TGF-β/BMP signaling cascades. In mouse embryonic stem cells (mESCs), TRIM33 regulates the transcription of marker genes involved in differentiation processes. Additionally, the association of TRIM33 with SMAD2 and SMAD3 has been shown to stimulate erythroid differentiation of hematopoietic stem/progenitor cells. These interactions highlight TRIM33's role as a context-dependent transcriptional regulator that responds to different cellular states during development .

What is the significance of TRIM33 co-localization with PML nuclear bodies?

TRIM33 demonstrates cell-context specific co-localization with Promyelocytic Leukemia Nuclear Bodies (PML NBs), which has significant implications for transcriptional regulation. Co-immunoprecipitation assays have confirmed that TRIM33 interacts with PML in various cell types, with notably stronger interactions observed in mouse embryonic stem cells (mESCs) compared to differentiated cells. This interaction is mediated by binding between their respective RBCC domains. The TRIM33-PML co-localization in mESCs forms clusters that regulate transcription of downstream target genes in the Nodal signaling pathway, particularly Lefty1/2, affecting stem cell differentiation and development. This cell-type specific localization pattern suggests that TRIM33 functions in context-dependent transcriptional regulatory centers that respond to different cellular states .

How can TRIM33 antibodies be used to investigate cancer biology?

TRIM33 antibodies provide valuable tools for investigating cancer biology through multiple approaches. Immunohistochemistry using TRIM33 antibodies has been validated in human lung cancer tissue and human brain tissue, allowing researchers to examine TRIM33 expression patterns in different tumor types. Western blot applications have been confirmed in several cancer cell lines including COLO 320, MCF-7, and PC-3, enabling quantitative analysis of TRIM33 expression levels across cancer models. Immunofluorescence analysis in MCF-7 cells can reveal subcellular localization patterns of TRIM33 in breast cancer cells. Since TRIM33 functions in cell proliferation control and participates in signaling pathways frequently dysregulated in cancer, these antibody applications can help elucidate TRIM33's role in oncogenesis, potentially identifying new therapeutic targets or diagnostic markers .

What factors might contribute to unexpected molecular weight observations for TRIM33 in Western blot?

When working with TRIM33 antibodies in Western blot applications, researchers might observe the protein at 140-150 kDa, which differs from the calculated molecular weight of 122 kDa. This discrepancy can be attributed to several factors: (1) Post-translational modifications, particularly ubiquitination and phosphorylation, which are relevant given TRIM33's function as an E3 ubiquitin ligase; (2) The presence of splice variants that may affect protein size; (3) The tripartite motif structure potentially affecting protein migration in SDS-PAGE; (4) Cell-type specific modifications that may vary between experimental systems. When troubleshooting unexpected bands, researchers should consider using positive controls (such as lysates from COLO 320, MCF-7, or PC-3 cells) where TRIM33 detection has been validated .

How can specificity of TRIM33 antibody be validated in experimental systems?

Validating TRIM33 antibody specificity is crucial for ensuring reliable experimental results. Multiple approaches should be employed: (1) Knockdown/knockout validation: At least one publication has confirmed antibody specificity using TRIM33 knockdown or knockout samples; (2) Multiple detection methods: Compare results across different techniques such as Western blot, immunoprecipitation, and immunofluorescence; (3) Positive controls: Use cell lines where TRIM33 expression has been confirmed, such as COLO 320, MCF-7, or PC-3 cells; (4) Peptide competition assays: Pre-incubating the antibody with the immunizing peptide should abolish specific signals; (5) Cross-reactivity testing: Though the antibody is designed for human TRIM33 detection, its reactivity with other species should be assessed if working in non-human models. These combined approaches ensure confidence in the antibody's specificity and experimental results .

What are the key considerations for quantitative analysis of TRIM33 expression using immunofluorescence?

When conducting quantitative analysis of TRIM33 expression using immunofluorescence, several methodological considerations are critical: (1) Fixation protocol: Optimize fixation conditions as over-fixation may mask epitopes while under-fixation can compromise cellular architecture; (2) Antibody dilution: For immunofluorescence applications, dilutions similar to those used in immunohistochemistry (1:50-1:500) may serve as starting points, but should be optimized; (3) Controls: Include secondary-only controls to assess background fluorescence and positive controls where TRIM33 localization is known; (4) Co-localization studies: When examining TRIM33 interaction with structures like PML nuclear bodies, appropriate co-staining markers should be included; (5) Quantification methods: Define clear parameters for measuring fluorescence intensity, counting positive cells, or assessing co-localization. The validated immunofluorescence protocol for MCF-7 cells using CSB-PA891796LA01HU at 1:100 dilution provides a good starting point for optimization .

How do different TRIM33 antibodies compare in terms of epitope recognition and application suitability?

Various commercially available TRIM33 antibodies target different epitopes and demonstrate different application profiles. The polyclonal antibody 55374-1-AP recognizes human TRIM33 and is validated for WB, IP, IF, IHC, and ELISA applications. The rabbit monoclonal antibody ab300146 (clone EPR25102-19) is validated specifically for WB and IP applications. The biotin-conjugated CSB-PA891796LD01HU polyclonal antibody is generated against recombinant human TRIM33 protein (amino acids 1-273) and is optimized for ELISA applications. When selecting between these antibodies, researchers should consider: (1) The specific application needs; (2) Whether polyclonal or monoclonal properties are preferred; (3) The detection system compatibility; (4) The specific epitope region being targeted, which may affect detection of splice variants or modified forms of TRIM33 .

How can TRIM33 research be integrated with studies of other TRIM family proteins?

Integrating TRIM33 research with studies of other TRIM family proteins provides valuable comparative insights into their shared and distinct functions. All TRIM family proteins contain the characteristic RBCC domain, which includes RING finger, B-box type zinc finger, and coiled-coil regions. When designing experiments to study multiple TRIM proteins: (1) Use antibodies validated for specificity to avoid cross-reactivity between family members; (2) Consider co-immunoprecipitation studies to examine potential interactions between TRIM33 and other TRIM proteins; (3) Employ comparative genomic approaches to understand evolutionary relationships; (4) Utilize proteomics to identify shared interacting partners or substrates; (5) Consider functional redundancy in knockout/knockdown studies. For instance, PML (also known as TRIM19) and TRIM33 have been shown to interact through their RBCC domains, suggesting functional relationships between different TRIM family members that may be explored using specific antibodies .

What methodological approaches can combine TRIM33 protein studies with transcriptional regulation analysis?

Investigating TRIM33's role in transcriptional regulation requires integrated approaches combining protein analysis with gene expression studies. Effective methodological strategies include: (1) Chromatin Immunoprecipitation (ChIP) using TRIM33 antibodies to identify genomic binding sites, which can be followed by sequencing (ChIP-seq) for genome-wide analysis; (2) RNA-seq following TRIM33 knockdown/knockout to identify regulated gene networks; (3) Co-immunoprecipitation with transcription factors or chromatin modifiers to map protein interaction networks; (4) Immunofluorescence co-localization studies with PML nuclear bodies or other transcriptional compartments; (5) Reporter gene assays to directly assess TRIM33's impact on target gene promoters, particularly in the TGF-β/BMP and Nodal signaling pathways. These combined approaches can elucidate how TRIM33 functions as a cell environment-dependent transcriptional regulatory center that responds to different cellular states and signals .