CDKN1C
Reactivity: Human
WB
Host: Rabbit
Polyclonal
unconjugated
Application Notes
Western Blot: 10 μg/mL for chemiluminescence detection system. Positive Controls: U937, MCF-7, ZR75-1, HL60. Detailed procedure is provided in Protocols. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user.
Protocol
SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mMTris-HCl, pH 7. 2, 250 mM NaCl, 0. 1% NP-40, 2 mM EDTA, 10% glycerol) containingappropriate protease inhibitors. Incubate it at 4°C with rotating for 30 minutes, thensonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C and transfer the supernatant toanother tube. Measure the protein concentration of the supernatant and add the Lysisbuffer to make 8 mg/mL solution. 3) Mix the sample with equal volume of Laemmli’s sample buffer. 4) Boil the samples for 2 minutes and centrifuge. Load 10 μL of the sample per lane in a 1mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hourin a semi-dry transfer system. (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for the transfer procedure. 6) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH7. 2) for 1 hour at room temperature, or overnight at 4°C. 7) Incubate the membrane with primary antibody diluted with PBS, pH 7. 2 containing 1%skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (Theoptimal antibody concentration will depend on the experimental conditions. 8) Wash the membrane with PBS (5 minutes x 6 times). 9) Incubate the membrane with the 1: 10000 POD-conjugated anti-mouse IgG diluted with1% skimmed milk (in PBS, pH 7. 2) for 1 hour at room temperature. 10) Wash the membrane with PBS (5 minutes x 6 times). 11) Wipe excess buffer from the membrane, then incubate it with appropriatechemiluminescence reagents for 1 minute. Remove extra reagent from the membrane bydabbing with a paper towel, and seal it in plastic wrap. 12) Expose to an X-ray film in a dark room for 5 minutes. Develop the film as usual. Theconditions for exposure and development may vary. Positive Controls for Western blotting: ZR75-1, HL60
Restrictions
For Research Use only
Concentration
1.0 mg/mL
Buffer
PBS, pH 7.2 containing 50 % Glycerol without preservatives.
Preservative
Without preservative
Storage
-20 °C
Storage Comment
Store the antibody undiluted at -20 °C. Shelf life: one year from despatch.
P57/Kip-2 (also known as CDKN 1C) is a potent inhibitor of several G1 cyclin/CDK2 complexes, and is a negative regulator of cell proliferation. It is closely-related to other cyclin dependent kinase inhibitors such as p21 (CIP1) and p27 (KIP-1), as they share a common N-terminal domain for binding to CDK/cyclin complexes and inhibiting their kinase activity. However, unlike Kip-1, Kip-2 is not regulated by p53. Kip-2 is involved in decisions to exit the cell cycle during development and differentiation, and overexpression of Kip-2 arrests cells in G1. The gene encoding human Kip 2 is located on chromosome 11p15.5, a region implicated in two sporadic cancers, Wilm's tumor and Beckwith Wiedemann syndrome (BWS, a cancer syndrome), making it a tumor suppressor candidate.Synonyms: Cyclin-dependent kinase inhibitor 1C, Cyclin-dependent kinase inhibitor p57, p57KIP2