p19 Skp1 (AA 4-158) antibody

Antigen: p19 Skp1

Binding Site: AA 4-158

Clonality: Monoclonal (52)

Host: Mouse

Reactivity: Human

Application: Western Blotting (WB), BioImaging (BI), Immunohistochemistry (IHC), Immunoprecipitation (IP)

Catalog no.: ABIN968019

Additional Information

Immunogen: Human p19 [Skp1]

Cross-Reactivity: Chicken, Dog (Canine), Frog, Mouse (Murine), Rat (Rattus)

Format: Liquid

Isotype: IgG1

Clone: 52

Description: In normal cells, Cyclin A and Cdk2 are components of an active quaternary complex of proteins that includes Cip1 (p21) and PCNA (proliferating cell nuclear antigen). This Cyclin A-Cdk2 complex is required during S phase. In transformed cells, Cyclin A and Cdk2 form complexes with three different proteins: p9, p19, and p45. p19 [Skp1], named for S phase kinase-dependent protein, and p45 [Skp2] are essential elements in the S phase kinase complex that is present in many transformed cell lines. p19 [Skp1] binds to Cyclin A-Cdk2 only when in combination with p45 [Skp2]. The mechanism that mediates p19 and p45 association with Cyclin A-Cdk2 is not yet known.

Characteristics: 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
6. Triton is a trademark of the Dow Chemical Company.

Molecular Weight: 19 kDa

Comments:

Related Products: ABIN967389, ABIN968533

Application Details

Application Notes: Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in an 96-well Imaging Plate and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 myl of Fixation Buffer to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either 90% methanol, or Triton™ X-100: a. Add 100 myl of -20°C 90% methanol to each well and incubate for 5 minutes at RT. OR b. Add 100 myl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 myl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 myl of to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 myl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 myl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 myl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 myl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 myl per well of 2 myg/ml Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.

Concentration: 250 µg/ml

Purity: Purified

Purification: Purified from tissue culture supernatant or ascites by affinity chromatography.

Buffer: Aqueous buffered solution containing BSA, glycerol.

Preservative: 0.09% Sodium azide.

Storage: Store undiluted at -20° C.

Restrictions: For Research Use only

Images

Western blot analysis of p19 [Skp1] on a A431 lysate. Lane 1: 1:5000, lane 2: 1:10000, lane 3: 1:20000 dilution of the p19 [Skp1] antibody.

Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate at ~ 10 000 cells per well. After overnight incubation, cells were stained using the alcohol perm protocol and the anti-p19 [Skp1] antibody. The second step reagent was FITC goat anti mouse Ig. The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells and can be used with either perm protocol.

anti-p19 Skp1 (AA 4-158) antibody (Image 3)

Publications

Product: Zhang, Kobayashi, Galaktionov et al.: "p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase." in: Cell, Vol. 82, Issue 6, pp. 915-25, 1995 (PubMed).

Liu, Kato, Zhang et al.: "beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, Issue 11, pp. 6273-8, 1999 (PubMed).

Carrano, Pagano: "Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression." in: The Journal of cell biology, Vol. 153, Issue 7, pp. 1381-90, 2001 (PubMed).

Saitoh, Pizzi, Wang: "Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358." in: The Journal of biological chemistry, Vol. 277, Issue 7, pp. 4755-63, 2002 (PubMed).