Overview

  • Product name

    Anti-p27 KIP 1 (phospho T187) antibody
    See all p27 KIP 1 primary antibodies
  • Description

    Rabbit polyclonal to p27 KIP 1 (phospho T187)
  • Host species

    Rabbit
  • Specificity

    ab75908 detects endogenous levels of p27 KIP 1 only when phosphorylated at threonine 187.
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human p27 KIP 1 conjugated to Keyhole Limpet Haemocyanin (KLH). Synthetic phosphopeptide derived from human p27 KIP 1 around the phosphorylation site of threonine 187 (E-Q-TP-P-K).
    Database link: P46527

  • Positive control

    • Extract from HeLa cells treated with EGF or IFN alpha. IF: A431 cell line

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Stable for 12 months at -20°C.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: 50% Glycerol, 0.87% Sodium chloride, PBS

    PBS without Mg2+ and Ca2+
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Purification notes

    ab75908 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.
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab75908 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/500 - 1/1000. Predicted molecular weight: 22 kDa.
IHC-P 1/50 - 1/100.
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function

    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.
  • Tissue specificity

    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.
  • Sequence similarities

    Belongs to the CDI family.
  • Domain

    A peptide sequence containing only AA 28-79 retains substantial Kip1 cyclin A/CDK2 inhibitory activity.
  • Post-translational
    modifications

    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.
  • Cellular localization

    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.
  • Information by UniProt
  • Database links

  • Alternative names

    • AA408329 antibody
    • AI843786 antibody
    • Cdki1b antibody
    • CDKN 1B antibody
    • CDKN 4 antibody
    • CDKN1B antibody
    • CDKN4 antibody
    • CDN1B_HUMAN antibody
    • Cyclin Dependent Kinase Inhibitor 1B antibody
    • Cyclin dependent kinase inhibitor p27 antibody
    • Cyclin-dependent kinase inhibitor 1B (p27, Kip1) antibody
    • Cyclin-dependent kinase inhibitor 1B antibody
    • Cyclin-dependent kinase inhibitor p27 antibody
    • Cyclin-dependent kinase inhibitor p27 Kip1 antibody
    • KIP 1 antibody
    • KIP1 antibody
    • MEN1B antibody
    • MEN4 antibody
    • OTTHUMP00000195098 antibody
    • OTTHUMP00000195099 antibody
    • p27 antibody
    • p27 Kip1 antibody
    • P27-like cyclin-dependent kinase inhibitor antibody
    • p27Kip1 antibody
    see all

Images

  • All lanes : Anti-p27 KIP 1 (phospho T187) antibody (ab75908) at 1/500 dilution

    Lane 1 : Extracts from HeLa cells treated with EGF with immunising peptide
    Lane 2 : Extracts from HeLa cells treated with EGF
    Lane 3 : Extracts from HeLa cells treated with IFN alpha

    Predicted band size: 22 kDa
    Observed band size: 27 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using ab75908 (left) at 1/50 dilution or the same antibody preincubated with blocking peptide (right).

  • ICC/IF image of ab75908 stained A431 cells (ab7909). The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75908, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:

  • Ale-Agha N  et al. CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine. PLoS Biol 16:e2004408 (2018). Read more (PubMed: 29927970) »
  • Zhang H & Hu N Telomerase reverse transcriptase induced thyroid carcinoma cell proliferation through PTEN/AKT signaling pathway. Mol Med Rep 18:1345-1352 (2018). Read more (PubMed: 29901196) »
See all 8 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (gradient 4-20%)
Sample
Mouse Cell lysate - whole cell (ACTH-secreting pituitary adenoma cell line)
Specification
ACTH-secreting pituitary adenoma cell line
Treatment
30 nM pre-miR-26a or 30 nM anti-miR-26a or specific oligo controls for 48hrs
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Dr. Erica Gentilin

Verified customer

Submitted Jul 17 2013

Answer

Thank you for getting back to me. Please find some details below regarding the protocol for cell treatment (with EGF or IFN-alpha). Method: 1). For adherent cells, cells were plated on flask or dishes containing growth medium (basic medium containing 10% serum). 2). When the cells were at the log phase and about 80-90% confluent, the culture media was drained. Wash cells with sterilized PBS three times. 3). Add fresh basic media to the vessels and place them to a CO2 incubator for starvation for 18-24 hours. 4). Drain the cultured medium, then add fresh basic medium (about 4 ml per T75 flask) to the vessels. Incubate the vessels to 37°C. 5). Add treating reagent to the medium at a certain concentration. 6). Cells were incubated at 37°C for needed time in incubator and then were used to prepare cell lysis. For suspension cells, when the cells were starved for 18-24 hours, cells were collected by centrifugation (1000g, 10 min) from cell supernatant. Resuspend the cell in 4 ml of fresh basic media per T75 flask. Others steps follow the 5th step above. Commonly used conditions of for stimulation: - EGF, 200ng/ml, 30 mins - IFN-alpha, 100ng/ml, 30 mins I hope this helps and if I can assist further, please do not hesitate to contact me.

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