EFTUD2 Antibody

What is EFTUD2 and why are antibodies against it valuable for research?

EFTUD2 (also known as SNU114, U5-116KD, or SNRP116) is a 116 kDa GTPase that functions as a core component of the spliceosome complex essential for pre-mRNA splicing. It plays crucial roles in mRNA maturation, immune response regulation, and has been implicated in viral infections and cancer progression . EFTUD2 antibodies enable researchers to detect this protein in various experimental contexts, allowing investigation of its expression, localization, and interactions. These antibodies are particularly valuable for studying RNA processing mechanisms, immune function regulation, and disease pathways where EFTUD2 has been implicated .

What applications have EFTUD2 antibodies been validated for?

EFTUD2 antibodies have been validated for multiple research applications:

Different antibodies may have specific optimal conditions, and it's recommended to titrate the antibody in each testing system to obtain optimal results .

How should EFTUD2 antibodies be stored and handled?

EFTUD2 antibodies are typically supplied in liquid form, often in PBS with preservatives such as 0.02% sodium azide and stabilizers like 50% glycerol and sometimes BSA . The recommended storage condition is -20°C, where they remain stable for approximately one year from receipt . After thawing, some antibodies can be stored at 2-8°C for up to 6 months . Avoiding repeated freeze-thaw cycles is crucial as this can degrade the antibody. For 20μl size preparations, some formulations contain 0.1% BSA as additional stabilizer . Always check manufacturer-specific recommendations for your particular antibody.

What species reactivity should be considered when selecting EFTUD2 antibodies?

When selecting EFTUD2 antibodies, consider that different products offer varying species reactivity profiles:

Species cross-reactivity should be experimentally verified for your specific research model . Some antibodies have demonstrated reactivity beyond what has been directly tested due to sequence homology, but validation in your species of interest is recommended.

How can I optimize Western blot conditions for EFTUD2 detection?

For optimal Western blot detection of EFTUD2:

• Gel selection: Use 7.5-10% SDS-PAGE gels for good resolution of this 116 kDa protein

• Sample preparation: HeLa or HEK293 cell lysates serve as reliable positive controls

• Loading amount: 15-50 μg of total protein per lane has been successfully used

• Antibody dilution: Start with 1:1000 and adjust based on signal strength

• Expected molecular weight: Look for bands at 116-122 kDa

• Controls: Consider EFTUD2 knockdown samples as negative controls, though complete knockout may be lethal in some cell types

• Detection system: Both chemiluminescence and fluorescence-based systems work well

Observe that some EFTUD2 antibodies may detect the protein at a slightly higher observed molecular weight (122 kDa) than the calculated weight (116 kDa) .

What considerations should be taken when using EFTUD2 antibodies for immunofluorescence studies?

For successful immunofluorescence with EFTUD2 antibodies:

• Cell selection: HepG2 cells have been validated for IF applications with EFTUD2 antibodies

• Fixation method: Both paraformaldehyde (4%) and methanol fixation can work; optimize for your specific antibody

• Antibody dilution: Start with 1:400-1:1600 as recommended

• Expected localization: Primarily nuclear staining where splicing occurs

• Validation: Use Iba1 co-staining when working with microglial cell models like BV2

• Controls: Consider EFTUD2 knockdown cells as negative controls

• Counter-staining: Nuclear staining (DAPI/Hoechst) helps confirm nuclear localization

Since EFTUD2 functions in pre-mRNA splicing, treatment of cells with splicing inhibitors prior to fixation can provide interesting insights and serve as functional controls.

What approaches can be used to study EFTUD2 in viral infection models?

EFTUD2 has demonstrated antiviral activity, particularly against HBV. To study this function:

• Cell models: Utilize HepG2.2.15 cells with integrated HBV genome

• Knockout/knockdown approach: Generate EFTUD2+/- cells using CRISPR-Cas9 or siRNA, confirming reduction via Western blot with EFTUD2 antibodies

• Viral parameters: Measure HBV DNA replication, HBeAg and HBsAg secretion, and HBcAg expression before and after manipulation of EFTUD2 levels

• Interferon treatment: Add IFN-α (5000 IU/mL has been used successfully) to examine EFTUD2's role in interferon-mediated antiviral responses

• RNA analysis: Perform RNA-seq to identify differentially expressed genes following EFTUD2 manipulation

• Protein analysis: Use EFTUD2 antibodies in combination with antibodies against ISGs (Mx1, OAS1, PKR) to assess downstream effects

These approaches have revealed that EFTUD2 promotes IFN-associated anti-HBV activity by regulating ISG expression through pre-mRNA splicing .

How can EFTUD2 antibodies be used to investigate its role in cancer biology?

EFTUD2 has been identified as a potential oncogene in hepatocellular carcinoma (HCC). To investigate its role:

• Expression analysis: Perform IHC with EFTUD2 antibodies on tissue microarrays comparing tumor and normal tissues

• Prognostic evaluation: Correlate EFTUD2 expression levels with patient survival and recurrence data

• Functional studies: Create knockdown/overexpression models and validate using EFTUD2 antibodies via Western blot

• Pathway analysis: Combine EFTUD2 antibodies with phospho-STAT3 antibodies to examine activation of oncogenic signaling

• Phenotypic assays: After confirming EFTUD2 modulation with antibodies, assess effects on cell viability, cell cycle progression, apoptosis, and metastatic potential

• Mechanistic investigation: Conduct RNA-seq followed by gene set enrichment analysis (GSEA) to identify pathways enriched in EFTUD2-overexpressing cells

What methodological approaches can uncover EFTUD2's role in splicing regulation?

To investigate EFTUD2's splicing regulatory functions:

• RNA immunoprecipitation: Use EFTUD2 antibodies to isolate associated RNA complexes, followed by sequencing to identify bound pre-mRNAs

• Splicing analysis: After EFTUD2 knockdown (confirmed with antibodies), perform RT-PCR or RNA-seq to identify differentially spliced transcripts

• Isoform quantification: Examine how EFTUD2 modulation affects the ratio of protein-coding versus non-coding transcript variants of target genes

• Rescue experiments: Perform overexpression of EFTUD2 in knockdown cells to restore splicing patterns, validating with EFTUD2 antibodies

• GTPase activity assessment: Create GTPase-deficient EFTUD2 mutants and compare splicing outcomes to wild-type, confirming expression with antibodies

Studies using these approaches have shown that EFTUD2 single allele knockout influenced the frequency of IFN-induced Mx1, OAS1, and PKR variant mRNA expressions, with protein-coding transcripts decreasing and non-protein coding transcripts increasing in EFTUD2+/- cells .

How do I investigate EFTUD2's role in immune regulation using antibodies?

EFTUD2 regulates immune responses through alternative splicing of key signaling components. To study this:

• Model selection: Use relevant immune cells (macrophages, BV2 microglial cells) or challenge hepatocytes with immune stimuli

• Knockdown approach: Transfect cells with EFTUD2 siRNA using Lipofectamine RNAimax for 3 days to ensure stable knockdown, confirming with antibodies

• Stimulation conditions: Challenge cells with inflammatory stimuli like LPS (1 μg/μL) for 12-24 hours

• Signaling pathway analysis: Examine how EFTUD2 modulation affects MyD88 and TLR signaling pathways using relevant antibodies

• ISG expression analysis: Measure expression of interferon-stimulated genes after EFTUD2 manipulation using both RNA and protein methods

• SNP analysis: Investigate how polymorphisms like rs3809756 in the EFTUD2 gene affect protein expression and function using genotype-specific samples

Research has shown that EFTUD2 regulates the innate immune response through alternative splicing of MyD88 and influences microglial activation in inflammatory conditions .

How can I validate the specificity of EFTUD2 antibodies?

To ensure EFTUD2 antibody specificity:

• Positive controls: Use HeLa, HEK293, or HepG2 cells which are known to express EFTUD2

• Negative controls: Implement EFTUD2 knockdown via siRNA (complete knockout may be lethal)

• Expected molecular weight: Confirm detection at the expected 116-122 kDa

• Blocking peptides: If available, pre-incubate antibody with the immunogen peptide to block specific binding

• Multiple antibodies: Use antibodies targeting different epitopes of EFTUD2 to confirm results

• Multiple techniques: Validate findings across different applications (WB, IF, IP)

• Cross-reactivity assessment: Test in cells from multiple species if working with non-human models

If working with developmental or neurological disorders associated with EFTUD2 mutations, consider comparing antibody reactivity between wild-type and mutant EFTUD2 proteins.

What are common challenges when working with EFTUD2 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with EFTUD2 antibodies:

• Lethality of complete knockout: Complete EFTUD2 knockout appears lethal in some cell types, so use conditional or inducible systems, or single allele knockout approaches

• Multiple isoforms: EFTUD2 has multiple transcript variants; verify which isoforms your antibody detects

• High molecular weight: The large size (116 kDa) requires longer transfer times in Western blotting and lower percentage gels

• Nuclear localization: For IF applications, ensure proper permeabilization to access nuclear EFTUD2

• Post-translational modifications: These may affect antibody recognition; consider phosphatase treatment if inconsistent results occur

• Batch-to-batch variation: Polyclonal antibodies may show variation; monoclonal alternatives like clone AB03/1B9 offer greater consistency

• Expression level differences: EFTUD2 expression may vary by cell type or condition; adjust antibody concentration accordingly

Always include appropriate controls and perform thorough validation before conducting critical experiments.

How should I interpret conflicting results when studying EFTUD2 in different experimental contexts?

When encountering conflicting results while studying EFTUD2:

• Antibody epitope comparison: Different antibodies target different regions (center region, AA 1-205, AA 257-284), potentially explaining discrepancies

• Cell type differences: EFTUD2 function may vary between cell types; HepG2.2.15 cells show different responses than neuronal or immune cells

• Condition-dependent effects: EFTUD2's role in viral infection or cancer may manifest differently depending on experimental conditions or disease stage

• Gene dosage effects: Single allele knockout (EFTUD2+/-) shows specific phenotypes that may differ from overexpression models

• Pathway interconnections: EFTUD2 affects multiple pathways (JAK/STAT, IFN response, EMT); context determines which predominates

• RNA vs. protein level discrepancies: Changes in splicing patterns may not always correlate with protein abundance

• Experimental timing: The temporal dynamics of EFTUD2 function, particularly in response to stimuli like IFN, may lead to different outcomes depending on assessment timing