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Purified Mouse Anti- p19 [Skp1]
Purified Mouse Anti- p19 [Skp1]

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.

Purified Mouse Anti- p19 [Skp1]

Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) 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 (Cat. No. 554001).  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 (see Recommended Assay Procedure).

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 (Cat. No. 353219) 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 (Cat. No. 554001).  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 (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog, Chicken, Frog (Tested in Development)
Mouse IgG1
Human p19 [Skp1] aa. 4-158
Western blot (Routinely Tested), Bioimaging, Immunohistochemistry, Immunoprecipitation (Tested During Development)
19 kDa
250 µg/ml
AB_397887
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging

1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.

2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well.  Incubate for 10 minutes at room temperature (RT).

3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:

a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

     OR

b. Add 100 μl 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 μl of 1× PBS.

5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.

6. Remove the blocking buffer and add 50 μl 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 μl of 1× PBS.

8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl 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 μl of 1× PBS.

10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11. View and analyze the cells on an appropriate imaging instrument.

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp

Western blot:  For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources 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.
610530 Rev. 3
Antibody Details
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52/p19 [Skp1]

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.

610530 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610530 Rev.3
Citations & References
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Development References (4)

  1. Carrano AC, Pagano M. Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression. J Cell Biol. 2001; 153(7):1381-1389. (Clone-specific: Western blot). View Reference
  2. Liu C, Kato Y, Zhang Z, Do VM, Yankner BA, He X. beta-Trcp couples beta-catenin phosphorylation-degradation and regulates Xenopus axis formation. Proc Natl Acad Sci U S A. 1999; 96(11):6273-6278. (Clone-specific: Western blot). View Reference
  3. Saitoh H, Pizzi MD, Wang J. Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358. J Biol Chem. 2002; 277(7):4755-4763. (Clone-specific: Immunofluorescence). View Reference
  4. Zhang H, Kobayashi R, Galaktionov K, Beach D. p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell. 1995; 82(6):915-925. (Biology). View Reference
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610530 Rev. 3

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.