Phospho-POLR2A CTD- S2/S5 Rabbit pAb - P24928

Core Product Specifications and Parameters

Biological Background: POLR2A Function and Localization

• DNA-directed RNA polymerase II subunit RPB1 is the catalytic core component of RNA polymerase II (Pol II), responsible for synthesizing mRNA precursors and many functional non-coding RNAs. Related references: PMID:23748380, PMID:27193682, PMID:30190596
• The Pol II transcription cycle proceeds through initiation, elongation, and termination stages, with the enzyme forming the active center together with the second largest subunit POLR2B/RPB2. Related references: PMID:23748380, PMID:27193682, PMID:28108474
• POLR2A appends nucleotides via a Mg(2+)-dependent mechanism and possesses a 3' -> 5' exonuclease proofreading activity to correct misincorporated ribonucleotides. Related references: PMID:30190596, PMID:8381534, PMID:9852112
• The unique C-terminal domain (CTD), consisting of 52 heptapeptide repeats, serves as a platform for transcription regulation; phosphorylation at Ser5 mediates promoter escape, while Ser2 phosphorylation is required for pause release and pre-mRNA processing. Related references: PMID:24207025, PMID:26124092
• CTD methylation and acetylation also regulate gene expression: acetylation is linked to immediate early gene induction, while methylation represses target gene expression.
• Subcellular localization includes nucleus, cytoplasm, and chromosome, consistent with its roles in transcription and RNA processing.
• POLR2A also functions as an RNA-dependent RNA polymerase, extending non-coding RNAs and acting as a replicase for Hepatitis delta virus.

Experimental Guidance and Technical Tips

• The immunogen is a synthetic peptide designed around the S2/S5 phosphorylation sites of the POLR2A CTD repeat YSPTSPS, suggesting the antibody may recognize both phosphorylated Ser2 and Ser5.
• For Western blot, due to the large molecular weight (~217 kDa), consider using low-percentage SDS-PAGE gels (e.g., 6-8%) and extended transfer times; inclusion of phosphatase inhibitors in lysis buffer is recommended to maintain phosphorylation.
• Specificity can be assessed by treating samples with lambda phosphatase or by peptide competition assays.
• In ELISA, use phospho-peptide coated plates and confirm reactivity with non-phospho peptide controls to evaluate phospho-selectivity.
• This antibody is indicated for human samples; cross-reactivity with other species has not been reported, so validation in the relevant sample system is advised.

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