sf3b3 Antibody

What is SF3B3 and why is it important in splicing research?

SF3B3 (Splicing Factor 3b Subunit 3) is a crucial component of the spliceosome, a large ribonucleoprotein complex responsible for removing introns from nuclear pre-mRNA through two-step transesterification reactions. SF3B3 functions as one of four proteins in the SF3B complex, which together with SF3A (composed of three proteins) forms the functional U2 small nuclear ribonucleoprotein (snRNP) . This protein is essential for 'A' complex assembly during splicing, specifically for the stable binding of U2 snRNP to the branchpoint sequence (BPS) in pre-mRNA .

SF3B3 has gained significant research attention not only for its fundamental role in RNA processing but also because of its involvement in various pathological conditions, particularly cancer progression. The protein has a calculated molecular weight of 136 kDa but is typically observed at 130-135 kDa in experimental settings .

How can I differentiate between SF3B3 and SAP130 in my research?

A critical consideration when working with SF3B3 antibodies is the historical confusion between SF3B3 and SAP130 (Sin3A associated protein 130). Despite being distinct proteins with different functions, SF3B3 was erroneously referred to as "spliceosome-associated protein 130" and abbreviated as "SAP130" in numerous publications . This ambiguity has led to significant experimental errors, including the use of incorrect antibodies and primers in SF3B3 research.

To differentiate between these proteins:

• Cellular localization: Immunohistochemical studies show that SF3B3 is predominantly nuclear, while SAP130 demonstrates a more ubiquitous distribution pattern .

• Function verification: SF3B3 functions in RNA splicing as part of the spliceosome, while SAP130 is a component of the histone deacetylase-dependent Sin3A corepressor complex .

• Antibody validation: Confirm antibody specificity through techniques like western blotting or immunoprecipitation followed by mass spectrometry to ensure you are studying the intended protein.

What applications are most suitable for SF3B3 antibodies?

SF3B3 antibodies have been successfully employed in various experimental applications, with immunofluorescence (IF) and immunocytochemistry (ICC) being particularly well-documented. Based on validation data:

• Immunofluorescence/ICC: SF3B3 antibodies have shown positive results in HepG2 and HeLa cells with recommended dilutions ranging from 1:50 to 1:500 .

• Immunohistochemistry (IHC): SF3B3 antibodies have been used in paraffin-embedded tissue sections with specific antigen retrieval protocols. The protocol typically involves de-paraffinization, antigen retrieval in Tris/EDTA buffer (pH 9.0) at 120°C for ten minutes, and overnight incubation with primary antibody at 4°C .

• Western blotting: Used to detect SF3B3 protein expression levels in tissue and cell lysates, with the protein typically observed at 130-135 kDa .

For optimal results, sample-dependent titration is recommended as antibody performance can vary based on experimental conditions and sample types .

What are the optimal storage conditions for SF3B3 antibodies?

For maximum stability and performance of SF3B3 antibodies:

• Storage temperature: Store at -20°C to maintain antibody integrity and activity .

• Light exposure: For fluorescent-conjugated antibodies like CoraLite® Plus 488-labeled SF3B3 antibodies (excitation/emission: 493 nm / 522 nm), avoid exposure to light to prevent photobleaching .

• Buffer composition: SF3B3 antibodies are typically stored in PBS with 50% glycerol, 0.05% Proclin300, and 0.5% BSA at pH 7.3 for optimal stability .

• Aliquoting considerations: While some antibody formulations may not require aliquoting for -20°C storage, dividing into single-use aliquots can prevent repeated freeze-thaw cycles that might compromise antibody quality .

• Shelf life: When stored under recommended conditions, SF3B3 antibodies remain stable for approximately one year after shipment .

How should SF3B3 antibodies be validated for cancer research applications?

Given SF3B3's emerging role in cancer biology, particularly colorectal cancer (CRC), rigorous antibody validation is essential:

• Expression comparison: Validate antibodies by comparing SF3B3 expression between cancer and adjacent normal tissues. Studies have demonstrated significantly elevated SF3B3 levels in CRC tissues (n=383) compared to surrounding normal tissues (n=51) .

• Knockout/knockdown controls: Generate SF3B3 knockdown cell lines (e.g., using specific siRNAs or shRNAs) as negative controls. Research has confirmed successful SF3B3 knockdown using two specific siRNAs, validated at both mRNA and protein levels .

• Rescue experiments: Perform rescue experiments by re-expressing SF3B3 in knockdown cells to confirm antibody specificity and functional effects. This approach has been successfully employed to demonstrate that SF3B3 knockdown effects on cell viability and colony formation could be largely rescued by SF3B3 re-expression .

• Cross-validation with multiple methods: Correlate antibody results with qRT-PCR data and western blot analysis to ensure consistency across detection methods .

• Tissue microarray analysis: For clinical samples, use tissue microarrays with paired cancer and normal tissues for standardized comparison and quantification of SF3B3 expression .

What methodological considerations are important when studying SF3B3's role in colorectal cancer progression?

Research has revealed that SF3B3 promotes colorectal cancer progression and metastasis through specific mechanisms. When designing experiments to investigate these processes:

• Functional assays selection: Include assays that assess both proliferation (cell viability, colony formation) and metastatic potential (migration, invasion assays) when studying SF3B3. Studies have shown that SF3B3 knockdown significantly suppresses cell viability, colony formation, migration, and invasion capacity of CRC cells .

• EMT marker assessment: Evaluate epithelial-mesenchymal transition (EMT) markers (E-cadherin, vimentin, N-cadherin) through western blot and immunofluorescence staining, as SF3B3 knockdown has been shown to enhance epithelial marker expression while reducing mesenchymal markers .

• In vivo model selection: Consider multiple in vivo models to comprehensively assess SF3B3's role:

  • Subcutaneous xenograft models for tumor growth

  • Tail vein injection models for lung metastasis

  • Orthotopic models (cecum termini injection) for primary tumor growth and liver metastasis evaluation

• Metastasis quantification: When assessing metastatic potential, quantify both the number and size of metastatic lesions, as both parameters have been shown to be affected by SF3B3 expression .

How can researchers accurately investigate SF3B3's molecular mechanisms in cancer?

To elucidate SF3B3's molecular functions in cancer:

• Pathway analysis approach: Perform KEGG pathway enrichment analysis of genes co-expressed with SF3B3. Studies have shown that SF3B3 positively co-expressed genes (1574 genes with |r|>0.2, p<0.05) are significantly enriched in the "spliceosome" pathway, while negatively co-expressed genes (993 genes) are predominantly enriched in the "metabolic" pathway .

• Alternative splicing analysis: Investigate SF3B3's role in regulating alternative splicing of specific targets like mTOR. Research has identified SF3B3 as a critical regulator of mTOR splicing and autophagy in multiple cancers, including CRC .

• Downstream signaling investigation: Examine SF3B3's impact on lipogenesis through the mTOR-SREBF1-FASN signaling axis, which has been identified as a key mechanism by which SF3B3 promotes CRC progression .

• Co-expression correlation: Analyze correlation between SF3B3 and proliferation markers like MKI67, which has demonstrated a positive correlation with SF3B3 expression .

What are the common pitfalls in SF3B3 antibody-based research and how can they be avoided?

Several critical errors have occurred in SF3B3 research due to confusion with SAP130:

• Antibody selection errors: Researchers have erroneously used antibodies against SAP130 instead of SF3B3. Always verify the target specificity of antibodies by checking the immunogen sequence and validation data .

• Primer design confusion: Some studies designed qPCR primers targeted at Sin3A associated protein 130 rather than SF3B3. Confirm primer specificity against the correct gene sequence (GenBank Accession Number: BC003146 for SF3B3) .

• Functional misattribution: SF3B3 has been incorrectly described as "a subunit of histone deacetylase" (which is SAP130's function). Review current literature on SF3B3's established role in RNA splicing .

• Incomplete methods reporting: Several publications have incomplete methods sections that fail to specify antibody sources and validation. Always provide complete antibody information including catalog numbers, RRID identifiers, and dilutions used .

• Lack of proper controls: Include appropriate positive and negative controls in all experiments, particularly when studying a protein with potential confusion in the literature.

What methodological approaches are recommended for studying SF3B3 in immunohistochemistry?

For optimal immunohistochemical detection of SF3B3:

• Tissue preparation: Section paraffin-embedded tissues at 4 μm thickness and mount on slides. De-paraffinize samples in a xylene and ethanol sequence .

• Endogenous peroxidase quenching: Incubate sections for 20 min in a solution of hydrogen peroxide methanol (0.1% H₂O₂ in MeOH) to reduce endogenous peroxidase activity .

• Antigen retrieval: Perform heat-induced epitope retrieval in Tris/EDTA buffer (pH 9.0) at 120°C for ten minutes, which has been validated for SF3B3 detection .

• Blocking and antibody incubation:

  • Block samples with PBT for 30 minutes

  • Incubate with primary anti-SF3B3 antibody overnight at 4°C (typical dilution ranges from 1:32,000 for high sensitivity antibodies to 1:100)

  • Incubate with appropriate secondary antibody (e.g., Poly-HRP-anti rabbit IgG) for 30 minutes

• Controls and localization expectations: Include appropriate controls and expect primarily nuclear localization for SF3B3, which helps distinguish it from the more ubiquitously distributed SAP130 .

What emerging research areas involve SF3B3 antibodies?

Based on current research trends: