The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 2 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
1/500 - 1/1000. Detects a band of approximately 59 kDa (predicted molecular weight: 59 kDa).
FunctionTyrosine protein phosphatase which functions as a dosage-dependent inducer of mitotic progression. Directly dephosphorylates CDK1 and stimulates its kinase activity. Also dephosphorylates CDK2 in complex with cyclin E, in vitro.
Sequence similaritiesBelongs to the MPI phosphatase family. Contains 1 rhodanese domain.
DomainThe phosphodegron motif mediates interaction with specific F-box proteins when phosphorylated. Putative phosphorylation sites at Ser-79 and Ser-82 appear to be essential for this interaction.
Post-translational modificationsPhosphorylated by CHEK1 on Ser-76, Ser-124, Ser-178, Ser-279, Ser-293 and Thr-507 during checkpoint mediated cell cycle arrest. Also phosphorylated by CHEK2 on Ser-124, Ser-279, and Ser-293 during checkpoint mediated cell cycle arrest. Phosphorylation on Ser-178 and Thr-507 creates binding sites for YWHAE/14-3-3 epsilon which inhibits CDC25A. Phosphorylation on Ser-76, Ser-124, Ser-178, Ser-279 and Ser-293 may also promote ubiquitin-dependent proteolysis of CDC25A by the SCF complex. Phosphorylation of CDC25A at Ser-76 by CHEK1 primes it for subsequent phosphorylation at Ser-79, Ser-82 and Ser-88 by NEK11. Phosphorylation by NEK11 is required for BTRC-mediated polyubiquitination and degradation. Phosphorylation by PIM1 leads to an increase in phosphatase activity. Phosphorylated by PLK3 following DNA damage, leading to promote its ubiquitination and degradation. Ubiquitinated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase complex that contains FZR1/CDH1 during G1 phase leading to its degradation by the proteasome. Ubiquitinated by a SCF complex containing BTRC and FBXW11 during S phase leading to its degradation by the proteasome. Deubiquitination by USP17L2/DUB3 leads to its stabilization.
Ab47400 staining human tonsil. Staining is localised to cytoplasm. Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
References for Anti-Cdc25A antibody (ab47400)
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