Purification notesThe antibody was affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific phosphopeptide. The antibody against non-phosphopeptide was removed by chromatography using non-phosphopeptide corresponding to the phosphorylation site.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/500 - 1/1000. Detects a band of approximately 130 kDa (predicted molecular weight: 107 kDa).
1/500 - 1/1000.
FunctionPleiotropic regulator of mitotic progression, participating in the control of spindle dynamics and chromosome separation. Phosphorylates different histones, myelin basic protein, beta-casein, and BICD2. Phosphorylates histone H3 on serine and threonine residues and beta-casein on serine residues. Important for G1/S transition and S phase progression.
Tissue specificityMost abundant in heart, liver, kidney and testis. Also expressed in smooth muscle cells and fibroblasts.
Sequence similaritiesBelongs to the protein kinase superfamily. NEK Ser/Thr protein kinase family. NIMA subfamily. Contains 1 protein kinase domain. Contains 6 RCC1 repeats.
Developmental stageExpression varied mildly across the cell cycle, with highest expression observed in G1 and stationary-phase cells.
DomainDimerizes through its coiled-coil domain.
Post-translational modificationsAutophosphorylated on serine and threonine residues. When complexed with FACT, exhibits markedly elevated phosphorylation on Thr-210. During mitosis, not phosphorylated on Thr-210. Phosphorylated by CDK1 in vitro.
Ab63553 staining Human colon. Staining is localised to the cytoplasm and nucleus. Left panel: with primary antibody at 1 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 AR 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-NEK9 (phospho T210) antibody (ab63553)
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