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Purified Mouse Anti-Human Cyclin A
Purified Mouse Anti-Human Cyclin A

Western blot analysis of Cyclin A on a A431 lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the Cyclin A antibody.

Purified Mouse Anti-Human Cyclin A

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-Cyclin A antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway™ 855 Bioimager system using a 20x objective.  This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells using both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Western blot analysis of Cyclin A on a A431 lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the Cyclin A 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-Cyclin A antibody.  The second step reagent was FITC goat anti mouse Ig (Cat. No. 554001).  The image was taken on a BD Pathway™ 855 Bioimager system using a 20x objective.  This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) cells using both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing)
Mouse IgG1
Human Cyclin A aa. 26-144
Western blot (Routinely Tested), Bioimaging (Tested During Development)
60 kDa
250 µg/ml
AB_398797
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.
611268 Rev. 3
Antibody Details
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25/Cyclin A

Progression of the mammalian cell cycle is regulated by phosphorylation of many key proteins. Several classes of cyclins (A-E) act as regulatory subunits for cyclin-dependent kinases (cdks). These cyclin-cdk holoenzymes are essential for proper control of cell cycle progression. They phosphorylate and regulate a variety of substrates whose activity is required for cell cycle transitions. The temporal expression of cyclins is tightly regulated throughout the cell cycle by synthesis and degradation. Such regulation plays a critical role in controlling the enzymatic activity of the cdks. Cyclin A, one of the mitotic cyclins, activates Cdk2 near the start of S phase and is necessary for the initiation of DNA replication. In mammalian somatic cells, Cyclin A is required during S phase and passage through G2. The D and E type cyclins regulate passage through G1, while Cyclin B is a critical regulator of mitosis. It has been shown in a number of species that mutation or disruption of normal Cyclin A expression causes cells to arrest at G2. Cyclin A binds both the cdc2 (Cdk1) and Cdk2 kinases and may also have a role in mitotic dependence on S phase completion.

611268 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
611268 Rev.3
Citations & References
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Development References (5)

  1. Giordano A, Whyte P, Harlow E, Franza BR Jr, Beach D, Draetta G. A 60 kd cdc2-associated polypeptide complexes with the E1A proteins in adenovirus-infected cells. Cell. 1989; 58(5):981-990. (Biology). View Reference
  2. Henglein B, Chenivesse X, Wang J, Eick D, Brechot C. Structure and cell cycle-regulated transcription of the human cyclin A gene. Proc Natl Acad Sci U S A. 1994; 91(12):5490-5494. (Biology). View Reference
  3. Pines J, Hunter T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J. 1994; 13(16):3772-3781. (Biology). View Reference
  4. Pines J. Cyclins and cyclin-dependent kinases: take your partners. Trends Biochem Sci. 1993; 18(6):195-197. (Biology). View Reference
  5. 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
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611268 Rev. 3

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