The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 0.5-1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use at 1-2 µg/mg of lysate.
Use a concentration of 0.5 - 1 µg/ml.
Use a concentration of 0.5 - 1 µg/ml.
Use a concentration of 0.5 - 1 µg/ml. Predicted molecular weight: 22 kDa.
Use a concentration of 0.5 - 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Important regulator of cell cycle progression. Involved in G1 arrest. Potent inhibitor of cyclin E- and cyclin A-CDK2 complexes. Forms a complex with cyclin type D-CDK4 complexes and is involved in the assembly, stability, and modulation of CCND1-CDK4 complex activation. Acts either as an inhibitor or an activator of cyclin type D-CDK4 complexes depending on its phosphorylation state and/or stoichometry.
Expressed in all tissues tested. Highest levels in skeletal muscle, lowest in liver and kidney.
Involvement in disease
Defects in CDKN1B are the cause of multiple endocrine neoplasia type 4 (MEN4) [MIM:610755]. Multiple endocrine neoplasia (MEN) syndromes are inherited cancer syndromes of the thyroid. MEN4 is a MEN-like syndrome with a phenotypic overlap of both MEN1 and MEN2.
Belongs to the CDI family.
A peptide sequence containing only AA 28-79 retains substantial Kip1 cyclin A/CDK2 inhibitory activity.
Phosphorylated; phosphorylation occurs on serine, threonine and tyrosine residues. Phosphorylation on Ser-10 is the major site of phosphorylation in resting cells, takes place at the G(0)-G(1) phase and leads to protein stability. Phosphorylation on other sites is greatly enhanced by mitogens, growth factors, cMYC and in certain cancer cell lines. The phosphorylated form found in the cytoplasm is inactivate. Phosphorylation on Thr-198 is required for interaction with 14-3-3 proteins. Phosphorylation on Thr-187, by CDK2 leads to protein ubiquitination and proteasomal degradation. Tyrosine phosphorylation promotes this process. Phosphorylation by PKB/AKT1 can be suppressed by LY294002, an inhibitor of the catalytic subunit of PI3K. Phosphorylation on Tyr-88 and Tyr-89 has no effect on binding CDK2, but is required for binding CDK4. Dephosphorylated on tyrosine residues by G-CSF. Ubiquitinated; in the cytoplasm by the KPC complex (composed of RNF123/KPC1 and UBAC1/KPC2) and, in the nucleus, by SCF(SKP2). The latter requires prior phosphorylation on Thr-187. Ubiquitinated; by a TRIM21-containing SCF(SKP2)-like complex; leads to its degradation. Subject to degradation in the lysosome. Interaction with SNX6 promotes lysosomal degradation.
Nucleus. Cytoplasm. Endosome. Nuclear and cytoplasmic in quiescent cells. AKT-or RSK-mediated phosphorylation on Thr-198, binds 14-3-3, translocates to the cytoplasm and promotes cell cycle progression. Mitogen-activated UHMK1 phosphorylation on Ser-10 also results in translocation to the cytoplasm and cell cycle progression. Phosphorylation on Ser-10 facilitates nuclear export. Translocates to the nucleus on phosphorylation of Tyr-88 and Tyr-89. Colocalizes at the endosome with SNX6 and this leads to lysosomal degradation.
Western blot - Anti-p27 KIP 1 antibody [SX53G8] (ab193379)
Predicted band size : 22 kDa
Lane 1: Wild-type HAP1 whole cell lysate (20 µg) Lane 2: CDKN1B knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg) Lane 4: MCF7 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab193379 observed at 30 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab193379 was shown to specifically recognize CDKN1B in wild-type HAP1 cells as well as additional cross-reactive bands. No bands were observed when CDKN1B knockout samples were used. Wild-type and CDKN1B knockout samples were subjected to SDS-PAGE. ab193379 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 µg/mL and 1/10,000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
Immunocytochemistry/ Immunofluorescence - Anti-p27 KIP 1 antibody [SX53G8] (ab193379)This image is courtesy of an Abreview submitted by Kirk Mcmanus
Ab193379 staining p27 KIP1 in HeLa cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100. Samples were incubated with primary antibody (1/200 in PBS) for 1 hour at 22°C. A Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) was used as the secondary antibody.