SCC4 Antibody

Basic Research Questions

How to validate SCC4 antibody specificity in experimental systems?

• Method: Perform Western blotting using cell lysates with known SCC4 expression levels (e.g., HeLa, MCF7) and include negative controls (knockdown or knockout cell lines). Compare observed molecular weight (~66–69 kDa) to predicted size (69 kDa) .

• Troubleshooting: Non-specific bands may arise due to cross-reactivity. Pre-absorb antibody with antigen-blocking peptides or use siRNA-mediated SCC4 knockdown to confirm specificity .

What experimental conditions optimize SCC4 detection in chromatin immunoprecipitation (ChIP)?

• Protocol: Crosslink cells with 1% formaldehyde, sonicate chromatin to 200–500 bp fragments, and use 1–2 μg antibody per IP. Validate enrichment at cohesin-loading sites (e.g., centromeres) via qPCR .

• Critical Controls: Include IgG isotype controls and target-negative genomic regions to assess background signal .

How to distinguish SCC4’s role in transcription regulation vs. cohesin loading?

• Approach: Combine RNA-seq (to assess transcriptional changes) with cohesin ChIP-seq in SCC4-depleted cells. Prioritize systems where SCC4’s dual roles are context-dependent (e.g., bacterial models vs. mammalian cells) .

Advanced Research Questions

How to resolve contradictions in SCC4 localization across cell cycle phases?

• Strategy: Use synchronized cell populations (e.g., double thymidine block) and perform immunofluorescence (IF) with anti-SCC4 antibody [EPR14390] at 1:100 dilution. Co-stain with cell cycle markers (e.g., Cyclin B1 for G2/M) .

• Data Interpretation: Note that SCC4 may dissociate from chromatin during mitosis, necessitating careful fixation/permeabilization (e.g., 0.1% Triton X-100) .

What methods confirm SCC4’s interaction with NIPBL/SCC2 in cohesin loading?

• Co-IP Workflow:

  • Lyse cells in mild detergent (e.g., 0.5% NP-40) to preserve protein complexes.

  • Immunoprecipitate SCC4 using 1–5 μg antibody [EPR14390] and detect NIPBL via Western blotting .

  • Validate with reciprocal IP (anti-NIPBL antibody) .

How to design multiplex assays integrating SCC4 antibody with T3SS effector studies?

• Experimental Design:

  • In C. trachomatis, co-express SCC4 with CopN and monitor secretion via SDS-PAGE/Western blot (use anti-CopN antibody) .

  • Pair with RNAP β-flap binding assays to dissect SCC4’s transcriptional vs. T3SS roles .

Table 1: SCC4 Antibody Performance Across Applications

Table 2: Common Technical Issues & Solutions

Methodological Insights

• Gene Expression vs. Cohesin Loading: To isolate SCC4’s transcriptional role (e.g., σ 66 RNAP interaction), use bacterial systems (E. coli) with IPTG-inducible SCC4 expression . For cohesin studies, employ mammalian cells with auxin-inducible SCC4 degradation .

• Quantitative Analysis: Normalize SCC4 ChIP-seq data to input DNA and use spike-in controls (e.g., Drosophila chromatin) for cross-experiment comparisons .