ZNF703 Antibody, HRP conjugated

How can researchers validate the specificity of ZNF703 antibodies in immunohistochemistry (IHC)?

Validation requires parallel testing with siRNA-mediated gene silencing and Western blot confirmation. In colorectal cancer (CRC) studies, researchers transfected LoVo cells with ZNF703-specific siRNA, observing >70% reduction in protein expression via Western blot . Subsequent IHC staining intensity decreased proportionally, confirming antibody specificity. Negative controls (omitting primary antibodies) must show no background staining . Cross-reactivity risks are minimized by selecting antibodies targeting unique epitopes, such as the N-terminal region (e.g., ARP39642_P050-HRP) .

What protocols optimize ZNF703 detection in Western blotting?

Protein extraction from nuclear and cytoplasmic fractions is critical, as ZNF703 localizes to both compartments . Use RIPA buffer with protease inhibitors and 30 µg of protein per lane. Primary antibody dilution at 1:1,000 with overnight incubation at 4°C yields optimal signal-to-noise ratios . For HRP-conjugated antibodies, enhance chemiluminescence substrates (e.g., ECL Western Blotting Substrate) improve sensitivity . Normalize using GAPDH or β-actin, and include cell lines with known ZNF703 expression levels (e.g., LoVo for high expression) .

How should ZNF703 expression scoring be standardized across tumor samples?

A semi-quantitative scoring system combines staining intensity (0–3) and percentage of positive cells (0–4), with final scores ranging from 0–12 . For example:

How can ZNF703 antibody elucidate mechanisms of immune checkpoint blockade resistance?

ZNF703 inversely correlates with CD8+ T cell infiltration and ICB response predictors (e.g., CD274/PD-L1) . In CRC cohorts, high ZNF703 expression associates with elevated TIDE scores, a marker of immune evasion . To investigate, co-stain tumor sections with ZNF703 and CD8 antibodies. Quantify spatial relationships using multiplex IHC or imaging mass cytometry. Functional validation requires CRISPR-Cas9 ZNF703 knockout in syngeneic mouse models treated with anti-PD-1 .

What strategies resolve contradictory findings in ZNF703’s role across cancer types?

In NSCLC, ZNF703 overexpression activates Akt/mTOR pathways, correlating with poor prognosis (p-Akt: 35.7% vs. 16.6% in low-ZNF703 tumors) . Conversely, in cholangiocarcinoma, ZNF703 silencing reduces migration by 60% (p<0.01) . To reconcile tissue-specific effects:

• Perform pan-cancer transcriptomic analyses (TCGA data) to identify co-expressed pathways.

• Use organoid models to test ZNF703 perturbations in defined microenvironments.

• Validate interactions via co-immunoprecipitation (e.g., ZNF703 binds HSP60/DCAF7 in breast cancer) .

How can ZNF703 antibody improve functional studies in 3D tumor models?

Incorporate HRP-conjugated antibodies into Matrigel-based invasion assays. Seed CRC cells (e.g., LoVo) in 8 μm Transwell inserts coated with Matrigel. After 24h, fix migrated cells and stain with ZNF703 antibody. Compare invasion rates between ZNF703-knockdown (siRNA) and overexpression (lentiviral) groups . For quantification, use automated cell counters or AI-based image analysis (e.g., ImageJ plugins).

What orthogonal methods confirm ZNF703 antibody specificity in chromatin studies?

Chromatin immunoprecipitation (ChIP) requires validation with:

• Negative controls: IgG isotype and no-antibody samples.

• Positive controls: Known ZNF703 targets (e.g., CCND1 in breast cancer) .

• CRISPR interference: dCas9-KRAB repression of ZNF703 loci reduces ChIP signal . Combine with RNA-seq to link binding events to transcriptional changes.