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Purified Mouse Anti-p27 [Kip1]
Purified Mouse Anti-p27 [Kip1]

Immunoprecipitation/Western Blot analysis of p27 [Kip1]. Lanes 1 and 2, Equal amounts of protein (25 µg/lane) from BALB/c 3T3 cell lysates of quiescent (lane 1) and proliferating (lane 2) cells were seperated by SDS-PAGE and were probed with clone G173-524 (Cat. No. 554069). Cells may be made quiescent by techniques such as serum starvation.  The antibody identifies p27 [Kip1] as a 27 kDa band and demonstrates that the level of p27 [Kip1] is higher during cell quiescence than during cell proliferation. Lane 3, lysate from quiescent BALB/c 3T3 cells was immunoprecipitated with clone G173-524. The immune complex was seperated by SDS-PAGE and p27 [Kip1] was detected by western blot analysis with clone G173-524.

Purified Mouse Anti-p27 [Kip1]

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-p27 [Kip1] antibody.  The second step reagent was Alexa Fluor® 555 goat anti-mouse IgG (Invitrogen).  The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective.  This antibody also stained A549 (ATCC CCL-185) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Immunoprecipitation/Western Blot analysis of p27 [Kip1]. Lanes 1 and 2, Equal amounts of protein (25 µg/lane) from BALB/c 3T3 cell lysates of quiescent (lane 1) and proliferating (lane 2) cells were seperated by SDS-PAGE and were probed with clone G173-524 (Cat. No. 554069). Cells may be made quiescent by techniques such as serum starvation.  The antibody identifies p27 [Kip1] as a 27 kDa band and demonstrates that the level of p27 [Kip1] is higher during cell quiescence than during cell proliferation. Lane 3, lysate from quiescent BALB/c 3T3 cells was immunoprecipitated with clone G173-524. The immune complex was seperated by SDS-PAGE and p27 [Kip1] was detected by western blot analysis with clone G173-524.

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-p27 [Kip1] antibody.  The second step reagent was Alexa Fluor® 555 goat anti-mouse IgG (Invitrogen).  The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective.  This antibody also stained A549 (ATCC CCL-185) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Product Details
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BD Pharmingen™
Mouse (QC Testing), Human (Tested in Development)
Mouse IgG1
Mouse p27 [Kip1] (full-length) Recombinant Protein
Western blot (Routinely Tested), Bioimaging, Immunoprecipitation (Tested During Development)
27 kDa
0.5 mg/ml
AB_395225
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°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. 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.
  4. 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.
  5. Triton is a trademark of the Dow Chemical Company.
554069 Rev. 9
Antibody Details
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G173-524

Cyclins and cyclin-dependent kinases (cdks) are evolutionarily conserved proteins that are essential for cell-cycle control in eukaryotes. Cyclins (regulatory subunits) bind to cdks (catalytic subunits) to form complexes that regulate the progression of the cell cycle.  These complexes are regulated by activating and inhibitory phosphorylation events, as well as by interactions with small proteins that bind to cyclins, cdks, or cyclin-cdk complexes. These include p15, p16, p18, p19, p21 and p27 [Kip1]. p27 [Kip1] has been shown to inhibit the activity of multiple cyclin-cdk complexes, including cyclin D-cdk4, cyclin E-cdk2 and cyclin A-cdk2. p27 [Kip1] is a 27 kD protein which shares N-terminal sequence homology with p21, and like p21, p27 [Kip1] contains a nuclear localization signal in its C-terminal region. IL-2 activation of T cells has been reported to lead to a decrease in p27 [Kip1] and entry into S phase. Removal of IL-2 from T cell cultures results in increased levels of p27 [Kip1] and cell quiescence.

554069 Rev. 9
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
554069 Rev.9
Citations & References
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Development References (6)

  1. Gorospe M, Liu Y, Xu Q, Chrest FJ, Holbrook NJ. Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2. Mol Cell Biol. 1996; 16(3):762-770. (Biology).
  2. Nourse J, Firpo E, Flanagan WM, et al. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994; 372(6506):570-573. (Biology). View Reference
  3. Polyak K, Kato JY, Solomon MJ, et al. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 1994; 8(1):9-22. (Biology). View Reference
  4. Polyak K, Lee MH, Erdjument-Bromage H, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell. 1994; 78(1):59-66. (Biology). View Reference
  5. Sherr CJ. Mammalian G1 cyclins. Cell. 1993; 73(6):1059-1065. (Biology). View Reference
  6. Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994; 78(1):67-74. (Biology). View Reference
View All (6) View Less
554069 Rev. 9

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