Phospho-POLR2A CTD-T4 Rabbit Polyclonal Antibody for WB, IP, ELISA, ChIP - P24928

Core Product Specifications and Parameters

Biological Background: POLR2A Function and Localization

• POLR2A encodes the largest subunit of RNA polymerase II (Pol II), a ~217 kDa protein that serves as the catalytic core for DNA-dependent RNA synthesis, essential for mRNA and non-coding RNA production. Related references: PMID:23748380, PMID:27193682, PMID:30190596
• The C-terminal domain (CTD) of POLR2A consists of 52 tandem heptapeptide repeats (YSPTSPS) that undergo dynamic phosphorylation, primarily at Ser2 and Ser5, to regulate transcription initiation, elongation, and termination, as well as co-transcriptional RNA processing. Related references: PMID:24207025, PMID:26124092
• POLR2A contains a DxDGD motif that coordinates Mg2+ ions essential for nucleotide addition, and also possesses a 3'-5' exonuclease activity for proofreading, ensuring high transcriptional fidelity. Related references: PMID:8381534, PMID:9852112
• Primarily localized in the nucleus and on chromosomes, POLR2A has also been detected in the cytoplasm, reflecting its dynamic shuttling and multifaceted roles.
• Post-translational modifications including serine/threonine phosphorylation, lysine acetylation, methylation, and ubiquitination modulate Pol II activity and stability.
• Alternative splicing generates multiple isoforms, and mutations in POLR2A are associated with neurodevelopmental disorders, highlighting its clinical significance.
• Also functions as an RNA-dependent RNA polymerase, extending non-coding RNAs, and acts as a replicase for Hepatitis delta virus during infection.

Experimental Guidance and Technical Tips

• Since the immunogen is a phospho-specific peptide corresponding to T4 of the POLR2A CTD repeats, validate antibody specificity by using non-phosphorylated peptide competition or phosphatase-treated cell lysates to confirm phospho-dependency.
• For Western blotting (WB), due to the large protein size (~217 kDa), consider using low-percentage SDS-PAGE gels (e.g., 6-8%) and extended transfer times to ensure efficient transfer of full-length POLR2A.
• The ChIP application may require optimization of crosslinking and chromatin shearing conditions; test different shearing methods (e.g., sonication or enzymatic digestion) to achieve chromatin fragments of 200–500 bp.
• In immunoprecipitation (IP) and ELISA experiments, include appropriate positive and negative controls, such as lysates from known POLR2A-expressing cell lines (e.g., HeLa, HEK293) and phospho-defective mutants if available.

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