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Purified Mouse Anti-Cdk8
Purified Mouse Anti-Cdk8

Western blot analysis of Cdk8 on a MDCK lysate (Canine Kidney; ATCC CCL-34). Lane 1: 0.5 µg/mL, lane 2: 0.25 µg/mL, and lane 3: 0.125 µg/mL of the Mouse Anti-Cdk8 antibody.

Purified Mouse Anti-Cdk8

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-Cdk8 antibody. The second step reagent was Alexa Fluor® 555 goat anti-mouse IgG (Invitrogen).  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 HRB-96) cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).

Western blot analysis of Cdk8 on a MDCK lysate (Canine Kidney; ATCC CCL-34). Lane 1: 0.5 µg/mL, lane 2: 0.25 µg/mL, and lane 3: 0.125 µg/mL of the Mouse Anti-Cdk8 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-Cdk8 antibody. The second step reagent was Alexa Fluor® 555 goat anti-mouse IgG (Invitrogen).  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 HRB-96) 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™
Dog (QC Testing), Human, Mouse, Chicken (Tested in Development)
Mouse IgG1
Human Cdk8 (C-terminal) Peptide
Western blot (Routinely Tested), Bioimaging (Tested During Development)
55 kD
None
AB_10053208
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.

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. 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.
  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. Triton is a trademark of the Dow Chemical Company.
  5. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
552053 Rev. 8
Antibody Details
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F9-1075

Cyclins, cyclin-dependent kinases (Cdks), and cyclin-dependent kinase inhibitors (CdkIs) are essential for cell-cycle control in eukaryotes. Cyclins, regulatory subunits, bind to cyclin-dependent kinases (Cdks), catalytic subunits, to form active cyclin-Cdk complexes. Cdk subunits by themselves are inactive and binding to a cyclin is required for their activity. Cyclins A, B1, D and E undergo periodic synthesis and degradation, thereby providing a mechanism to regulate Cdk activity throughout the cell cycle.  In contrast to other members of the cyclin family, cyclin C does not have a role in regulating cell cycle progression. Cyclin C and its partner, Cdk8 are associated with RNA polymerase II, an enzyme responsible for transcribing protein encoding genes. Research has shown that Cdk8 stabilizes cyclin C to prevent its degradation, moreover this stabilization exists for either the catalytically active or inactive form of Cdk8. This is in agreement with previous work showing that the kinase activity of the cyclin C - Cdk8 does not change during the cell cycle.

552053 Rev. 8
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
552053 Rev.8
Citations & References
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Development References (3)

  1. Barette C, Jariel-Encontre I, Piechaczyk M, Piette J. Human cyclin C protein is stabilized by its associated kinase cdk8, independently of its catalytic activity. Oncogene. 2001; 20(5):551-562. (Biology). View Reference
  2. Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Annu Rev Pharmacol Toxicol. 1999; 39:295-312. (Biology). View Reference
  3. Rickert P, Corden JL, Lees E. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene. 1999; 18(4):1093-1102. (Biology). View Reference
552053 Rev. 8

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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.