UTP11 Antibody, FITC conjugated

How can researchers validate the specificity of UTP11 Antibody, FITC conjugated in immunofluorescence assays?

Validation of antibody specificity requires a multi-step approach. First, perform siRNA-mediated knockdown of UTP11 in target cell lines (e.g., HCT116 or HepG2) and confirm reduced protein levels via Western blot . For immunofluorescence, use cells transfected with UTP11-targeting siRNA alongside non-targeting controls. A validated antibody should show diminished fluorescence signal in knockdown cells compared to controls. Additionally, cross-verify localization patterns with subcellular fractionation assays; UTP11 is nucleolar, so co-staining with nucleolar markers like NPM1/B23 is critical . Include secondary antibody-only controls to exclude autofluorescence artifacts. If working with FFPE tissues, antigen retrieval optimization (e.g., pH 9.0 Tris-EDTA buffer) is essential to expose epitopes masked during fixation .

What experimental parameters optimize Western blot detection of UTP11 using FITC-conjugated antibodies?

Optimal Western blot conditions involve:

• Lysis Buffer: Use RIPA buffer supplemented with RNase inhibitors (e.g., SUPERase-In™) to preserve RNA-protein complexes .

• Gel Electrophoresis: 4–12% Bis-Tris gels under reducing conditions (50 mM DTT) to resolve UTP11’s ~55 kDa band .

• Transfer: Semi-dry transfer at 25 V for 1 hour to nitrocellulose membranes (0.45 μm pore size).

• Blocking: 5% BSA in TBST to minimize non-specific binding.

• Antibody Dilution: 1:500–1:1,000 dilution in blocking buffer, incubated overnight at 4°C .

• Detection: FITC fluorescence requires imaging systems like LI-COR Odyssey® with 488 nm excitation. Normalize signals to vinculin or GAPDH .

What controls are necessary for UTP11 antibody validation in immunohistochemistry (IHC)?

Critical controls include:

• Isotype Control: Same-species IgG conjugated to FITC at matching concentrations.

• Tissue Specificity: Compare HCC tumors (high UTP11 expression) to adjacent normal liver tissues .

• Preabsorption Control: Preincubate antibody with a 10-fold molar excess of recombinant UTP11 protein; signal should diminish.

• Disease Correlation: Validate staining against TCGA data showing UTP11 overexpression in HCC (AUC = 0.894) .

How can UTP11 Antibody, FITC conjugated be applied to study RNA-protein interactions via RNA immunoprecipitation (RIP)?

To investigate UTP11’s role in mRNA stabilization (e.g., NRF2), perform RIP as follows :

• Crosslinking: Treat cells with 1% formaldehyde for 10 min to fix RNA-protein interactions.

• Lysis: Use RIP buffer (10 mM Tris, 150 mM NaCl, 0.5% NP-40) with RNase inhibitors.

• Immunoprecipitation: Incubate lysates with FITC-conjugated UTP11 antibody-bound magnetic beads overnight at 4°C.

• RNA Isolation: Purify co-precipitated RNA using TRIzol® and analyze targets (e.g., NRF2, Oct4) via RT-qPCR. Include input (5% lysate) and IgG controls. A 5-fold enrichment of target mRNA in IP vs. IgG confirms specificity .

What methodological strategies resolve discrepancies in UTP11 localization across cancer models?

Discrepancies in nucleolar vs. cytoplasmic localization often arise from:

• Cell Line Variability: HCT116 cells show strong nucleolar UTP11, while HepG2 may exhibit cytoplasmic leakage under stress . Validate using nucleolar isolation kits (e.g., Nucleolar Protein Isolation Kit, Sigma).

• Fixation Artifacts: Methanol fixation (−20°C, 10 min) preserves nucleolar architecture better than paraformaldehyde .

• Stress Conditions: Ribosomal stress (e.g., Actinomycin D) redistributes UTP11. Correlate localization with nucleolar stress markers (RPL5/RPL11) .

How to design experiments analyzing UTP11’s dual role in p53 activation and ferroptosis?

Experimental Workflow:

• Genetic Models: Use isogenic p53+/+ and p53−/− HCT116 cells .

• UTP11 Knockdown: Transfect siRNA/shRNA and confirm efficiency via qRT-PCR (primers: F-5′-CAGGTCAACGGATTTGGTCT-3′, R-5′-GCAAGTTCCGTTCTCAGTCC-3′) .

• Phenotypic Assays:

  • p53 Activity: Measure p21 and MDM2 ubiquitination via cycloheximide chase assays .

  • Ferroptosis: Quantify lipid peroxidation (MDA assay) and GSH/GSSG ratios .

• Cross-talk Analysis: Inhibit p53 (Pifithrin-α) and assess residual ferroptosis to isolate p53-independent effects .

How to reconcile conflicting reports on UTP11’s prognostic value in hepatocellular carcinoma?

Key Variables:

Resolution:

• Cohort Heterogeneity: TCGA data reflect bulk tumors, obscuring intratumoral UTP11 variability. Single-cell RNA-seq is recommended.

• Antibody Clones: Commercial UTP11 antibodies (e.g., Signalway #46701 vs. Santa Cruz sc-271737) target distinct epitopes. Validate using CRISPR-Cas9 KO lines .