Anti-HpTGEKP motif Antibody

C2H2 zinc finger proteins (ZFPs) represent the largest family of gene regulators in human. C2H2 ZFPs wrap around DNA via multiple zinc fingers to ensure a stable DNA interaction. Small linker peptides joining adjacent zinc finger modules are critical for the DNA-binding activity of C2H2 ZFPs. The vast majority of linker peptides contain a serine or threonine as their first amino acid residue, phosphorylation of which greatly reduces the DNA binding activity of C2H2 ZFPs. TGEKP is the most common consensus linker sequence found in C2H2 ZFPs, including YY1, Aiolos, TIP20, and Bcl6. Antibody-based detection of H-pTGEKP (H represents the His residue from the preceding zinc finger, and pT represents phospho-Thr) reveals a large scale and tightly synchronized mitotic phosphorylation of ZFPs, starting in mid-prophase right after DNA condensation and before the breakdown of the nuclear envelope. T-lymphokine-activated killer cell-originated protein kinase (TOPK; aka PBK) is activated by Cdk1 and is identified as the kinase responsible for the inactivation of C2H2 ZFPs during mitotic cell division by phosphorylating the TGEKP linker sequence. TOPK/PBK is upregulated in many human cancers and its high expression correlates with poor prognosis.

Various molecular weights due to presence of conserved motif in multiple proteins.

This antibody recognizes mulitple phosphorylated mitotic proteins.

KLH-conjugated linear peptide corresponding to the HpTGEKP motif.

Anti-HpTGEKP motif Antibody is a Rabbit Polyclonal Antibody for detection of HpTGEKP motif also known as HpTGEKP, global mitotic phosphorylation marker & has been validated in WB, ICC, FC & IP.

Western Blotting Analysis: A representative lot detected TOPK-catalyzed phosphorylation of Aiolos, BCL6, TIP20, and YY1 C2H2 linker sequences in in vitro kinase assays using GST fusion constructs containing the respective linker sequences (Rizkallah, R., et al. (2014). Oncotarget. In press).

Immunocytochemistry Analysis: A representative lot was used in combination with an anti-pTOPK antibody for a dual immunofluorescent staining of synchronized HeLa cells at different time points after thymidine block release. Spatiotemporal correlation between the mitotic activation of TOPK and C2H2 linker phosphorylation is consistent with TOPK′s role as the linker kinase (Rizkallah, R., et al. (2014). Oncotarget. In press).

Immunocytochemistry Analysis: A 1:500 dilution from a representative lot was employed to detect C2H2 linker phosphorylation in HeLa cells following double-thymidine block and release (Rizkallah, R., et al. (2011). Cell Cycle. 10(19):3327-3336).

Flow Cytometry Analysis: A representative lot was employed to detect C2H2 linker phosphorylation in asynchronous (Asy) and thymidine-arrested (Thy) HeLa cells. (Rizkallah, R., et al. (2011). Cell Cycle. 10(19):3327-3336).

Immunoprecipitation Analysis: A representative lot immunoprecipitated C2H2 ZFPs with phosphorylated pTGEKP linker sequence from pro-metaphase HeLa cells following nocodazole treatment.

Control
Untreated and Nocodazole treated HeLa acid extracts

Evaluated by Western Blot in untreated and nocodazole treated HeLa acid extracts.

Western Blot Analysis: 1 µg/mL of this antibody detected C2H2 ZFPs with phosphorylated pTGEKP linker sequence in acid extracts from nocodazole-treated, but not untreated HeLa cells.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.