TRIM26 Antibody

What is TRIM26 and why is it significant in immunological research?

TRIM26 (Tripartite Motif Containing 26) is an E3 ubiquitin ligase encoded within the major histocompatibility complex class I region. It plays a critical role in inflammatory immune responses by positively regulating TAK1-mediated NF-κB activation through K11-linked polyubiquitination of TAB1. TRIM26 expression increases following stimulation with various TLR ligands (LPS, Pam3CSK4, Poly(I:C), R848) and proinflammatory cytokines (TNF-α, IL-1β), suggesting its importance in inflammation regulation . Recent screening has also identified TRIM26 as a critical host factor for HCV replication .

What are the recommended applications for TRIM26 antibodies?

TRIM26 antibodies have been validated for multiple research applications:

How should I validate TRIM26 antibody specificity?

Validation of TRIM26 antibody specificity is essential to ensure reliable results. Implement the following methodological approaches:

• Compare signal between wild-type and TRIM26 knockout or knockdown samples

• Utilize CRISPR-Cas9 generated TRIM26 knockout cell lines, as described in published protocols

• Perform siRNA knockdown experiments using TRIM26-specific siRNAs

• Test antibody reactivity with overexpressed TRIM26 in transfection experiments

• Include appropriate positive controls (cells known to express TRIM26, such as HEK293T) and negative controls (TRIM26-deficient cells)

What is the optimal protocol for detecting TRIM26 by Western blot?

For optimal Western blot detection of TRIM26:

• Lyse cells in RIPA buffer supplemented with protease inhibitors

• Separate 20-50 μg total protein on 10% SDS-PAGE gels

• Transfer to PVDF membrane (recommended over nitrocellulose)

• Block with 5% non-fat milk or BSA in TBST

• Incubate with TRIM26 antibody (1:1000-1:2000) overnight at 4°C

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody

• Develop using ECL detection system

For enhanced specificity, consider using TRIM26 knockout/knockdown samples as negative controls .

How can I design experiments to study TRIM26's role in inflammatory pathways?

When investigating TRIM26's role in inflammatory signaling:

• Cell preparation: Use primary cells (peritoneal macrophages, BMDMs) or cell lines from both wild-type and TRIM26-deficient sources

• Stimulation: Treat cells with TLR ligands (LPS, Pam3CSK4, Poly(I:C), R848) or proinflammatory cytokines (TNF-α, IL-1β) for various time points (0-24h)

• Pathway analysis: Examine phosphorylation of IKKα/β, p65, JNK, p38, and ERK using phospho-specific antibodies

• Cytokine production: Measure mRNA expression (qPCR) and protein secretion (ELISA) of TNF-α, IL-6, and IL-12p40

• Ubiquitination assays: Immunoprecipitate TAB1 and blot with anti-ubiquitin antibodies (preferably K11-linkage specific)

How should I optimize immunoprecipitation experiments with TRIM26 antibody?

For successful TRIM26 immunoprecipitation:

• Use mild lysis buffers (e.g., 1% NP-40 or 0.5% Triton X-100) to preserve protein interactions

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Use 2-5 μg of TRIM26 antibody per 500-1000 μg protein lysate

• Include appropriate controls (IgG control, TRIM26-deficient samples)

• For ubiquitination studies, add deubiquitinase inhibitors (10 mM N-Ethylmaleimide) and proteasome inhibitors (10 μM MG132) to lysis buffer

• When studying TRIM26-TAB1 interactions, consider reverse co-IP (immunoprecipitate with TAB1 antibody and detect TRIM26)

How can I study TRIM26-mediated ubiquitination of target proteins?

To investigate TRIM26's E3 ligase activity and ubiquitination of targets such as TAB1:

• Cell-based ubiquitination assay:

  • Transfect cells with constructs expressing tagged versions of TRIM26, target protein (e.g., TAB1), and ubiquitin (HA-Ub)

  • Immunoprecipitate the target protein (e.g., Myc-TAB1)

  • Detect ubiquitination by immunoblotting with anti-ubiquitin or anti-tag antibody

  • Include ubiquitin mutants (e.g., K11-only, K11R) to determine linkage specificity

• In vitro ubiquitination assay:

  • Use recombinant TRIM26 and target proteins prepared with TNT Quick Coupled Transcription/Translation System

  • Add E1, E2 enzymes, ATP, and ubiquitin

  • Detect ubiquitination by Western blotting

• Endogenous ubiquitination:

  • Stimulate cells with appropriate ligands (e.g., LPS)

  • Immunoprecipitate endogenous target protein (e.g., TAB1)

  • Detect ubiquitination using anti-ubiquitin antibodies

How do I analyze contradictory results when studying TRIM26 in different cell types?

When facing contradictory results across different cell types:

• Verify endogenous expression levels: Different cell types may express varying levels of TRIM26 and its interacting partners

• Check for cell-specific post-translational modifications: TRIM26 function may be regulated differently

• Examine expression of other TRIM family members: Functional redundancy might explain differences

• Confirm antibody specificity in each cell type: Some antibodies may cross-react with related proteins

• Consider genetic approaches: Generate TRIM26 knockout in multiple cell types using CRISPR-Cas9

• Perform rescue experiments: Re-express TRIM26 in knockout cells to confirm observed phenotypes

• Compare primary cells vs. cell lines: The search results show differences between primary macrophages and established cell lines

What controls are essential when studying TRIM26 knockout models?

When working with TRIM26 knockout models:

• Genotyping confirmation: Verify deletions/mutations by PCR and sequencing as described in the TALEN-generated knockout mice

• Protein expression validation: Confirm absence of TRIM26 protein by Western blot

• Functional complementation: Re-express wild-type TRIM26 to restore function

• Domain mutant controls: Express catalytically inactive TRIM26 (e.g., C16A mutant mentioned in methodology)

• Littermate controls: Use age-matched, same-sex littermates for in vivo experiments

• Pathway validation: Confirm expected changes in NF-κB and MAPK signaling pathways

• Phenotypic validation: Verify inflammatory phenotypes (e.g., cytokine production) match published results

How can TRIM26 antibodies be used to investigate virus-host interactions?

For studying TRIM26's role in viral infections:

• Infection models: Use TRIM26 knockout or knockdown cell lines alongside wild-type controls for viral infection studies

• Time-course experiments: Monitor TRIM26 expression changes during infection

• Colocalization studies: Perform immunofluorescence to detect TRIM26 and viral proteins

• Protein-protein interactions: Use co-immunoprecipitation to identify virus-TRIM26 interactions

• Viral replication assays: Measure viral replication markers (e.g., luciferase reporters for HCV)

• Rescue experiments: Reintroduce wild-type or mutant TRIM26 to determine functional domains important for viral replication

What are the best approaches for studying TRIM26 in primary immune cells?

For investigating TRIM26 in primary immune cells:

• Isolation protocols: Follow established protocols for isolating peritoneal macrophages, BMDMs, or MEFs from wild-type and TRIM26-deficient mice

• Stimulation conditions: Stimulate cells with appropriate TLR ligands or cytokines at optimal concentrations (e.g., LPS 200 ng/ml)

• Time-course analysis: Monitor TRIM26 expression and downstream signaling at multiple time points

• Ex vivo culture optimization: Maintain appropriate culture conditions (DMEM with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 37°C, 5% CO2)

• Genetic manipulation: Use lentiviral vectors for gene knockdown or overexpression in primary cells

• Functional readouts: Measure cytokine production, phosphorylation of signaling molecules, and ubiquitination of target proteins

How should TRIM26 antibodies be validated for chromatin immunoprecipitation studies?

For ChIP applications with TRIM26 antibodies:

• Antibody selection: Choose ChIP-grade antibodies specifically validated for this application

• Pre-clearing optimization: Minimize background by optimizing pre-clearing steps

• Crosslinking conditions: Test different formaldehyde concentrations and incubation times

• Sonication optimization: Adjust sonication parameters to achieve 200-500 bp chromatin fragments

• Controls: Include IgG control and TRIM26-deficient samples

• Sequential ChIP: Consider sequential ChIP (re-ChIP) to identify co-occupancy with other transcription factors

• Validation by qPCR: Confirm enrichment at candidate regions before proceeding to sequencing

How can I resolve issues with non-specific bands when using TRIM26 antibodies?

When encountering non-specific bands:

• Optimize blocking conditions: Test different blocking agents (milk, BSA, commercial blockers)

• Adjust antibody concentration: Titrate primary antibody to find optimal dilution

• Increase washing stringency: Use higher salt concentrations or add mild detergents to wash buffers

• Compare multiple antibodies: Test antibodies targeting different epitopes of TRIM26

• Use genetic controls: Compare with TRIM26 knockout or knockdown samples to identify specific bands

• Pre-absorb antibody: Incubate with TRIM26-deficient lysate to reduce non-specific binding

• Optimize protein extraction: Test different lysis buffers to improve specificity

What are common pitfalls when studying TRIM26 in ubiquitination assays?

Common challenges in TRIM26 ubiquitination studies include:

• Transient ubiquitination: Add deubiquitinase inhibitors and proteasome inhibitors to preserve ubiquitinated species

• Linkage specificity: Use linkage-specific antibodies (K11-linked) for more precise analysis

• Background ubiquitination: Include appropriate controls (inactive TRIM26 mutants)

• E2 enzyme specificity: Different E2 enzymes can affect the type of ubiquitin chains formed

• Substrate specificity: TRIM26 catalyzes K11-linked polyubiquitination of TAB1 at specific lysine residues (Lys294, Lys319, and Lys335), so mutations in these residues may affect results

• Interference from other E3 ligases: RNF114 also ubiquitinates TAB1, though through different mechanisms

• Validation in multiple systems: Confirm findings in both cell-based and in vitro assays

How should I quantify and normalize TRIM26 expression data across different experimental conditions?

For accurate quantification and normalization:

• Loading controls: Use housekeeping proteins (β-actin, GAPDH) consistently across experiments

• Densitometry: Utilize image analysis software for quantification of Western blot bands

• Normalization strategy: Normalize TRIM26 signal to loading control for each sample

• Multiple time points: Capture dynamic changes in TRIM26 expression following stimulation

• Statistical analysis: Apply appropriate statistical tests to determine significance

• Biological replicates: Perform at least three independent experiments

• Cross-methodology validation: Confirm protein expression changes with mRNA analysis (qPCR)