Immunofluorescent staining of A549 (ATCC CCL-185) 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 Triton™ X-100 perm protocol and the anti-KAP 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 HeLa (ATCC CCL-2) and U-2 OS (ATCC HTB-96) cells and can be used with either fix/perm protocol (see Recommended Assay Procedure).
Immunofluorescent staining of A549 (ATCC CCL-185) 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 Triton™ X-100 perm protocol and the anti-KAP 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 HeLa (ATCC CCL-2) and U-2 OS (ATCC HTB-96) cells and can be used with either fix/perm protocol (see Recommended Assay Procedure).
Preparation And Storage
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
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
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Proteins that associate with cyclin-dependent kinases may function in cell cycle regulation. Therefore, a two-hybrid screen system was used to isolate cDNA which encode proteins that can interact with Cdk2. One of these was the dual-specificity protein phosphatase known as KAP (Cdk associated phosphatase). KAP contains the HCXXXXGR motif characteristic of protein tyrosine phosphatases. It utilizes substrates that contain either phosphotyrosine or phosphoserine residues. In addition to binding Cdk2, KAP also associates with Cdc2. KAP does not require a cyclin subunit for binding to Cdk2 and Cdc2. The association of KAP with these two Cdks suggests that KAP may play a role in cell cycle control, possibly by regulating the phosphorylation status of a cdk or of a Cdk-associated protein.
Development References (2)
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Hannon GJ, Casso D, Beach D. KAP: a dual specificity phosphatase that interacts with cyclin-dependent kinases. Proc Natl Acad Sci U S A. 1994; 91(5):1731-1735. (Biology). View Reference
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Lee SW, Reimer CL, Fang L, Iruela-Arispe ML, Aaronson SA. Overexpression of kinase-associated phosphatase (KAP) in breast and prostate cancer and inhibition of the transformed phenotype by antisense KAP expression. Mol Cell Biol. 2000; 20(5):1723-1732. (Clone-specific: Immunofluorescence, Immunohistochemistry, Western blot). View Reference
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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.
