Purified Mouse Anti- PKA [RI]
Clone 18/PKA [RI] (RUO)
- Brand BD Transduction Laboratories™
- Concentration 250 µg/ml
- Isotype Mouse IgG2b
- Reactivity Human (QC Testing) Mouse, Rat, Dog, Chicken, Frog (Tested in Development)
Western blot (Routinely Tested)
Bioimaging, Immunoprecipitation (Tested During Development)
- Immunogen Mouse PKA [RI] subunit aa. 225-381
- Storage Buffer Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
cAMP-dependent Protein Kinase (PKA) is composed of two distinct subunits: catalytic (C) and regulatory (R). Four regulatory subunits have been identified: RIα, RIß, RIIα, and RIIß. These subunits define type I and II cAMP-dependent protein kinases. Following binding of cAMP, the regulatory subunits dissociate from the catalytic subunits, rendering the enzyme active. Type I and type II holoenzymes have three potential C subunits (Cα, Cß, or Cγ). Type II PKA can be distinguished by autophosphorylation of the R-subunits, while type I PKA binds Mg/ATP with high affinity. Most cells express both type I and type II PKAs. Although the Rα isoforms are ubiquitously expressed, the Rß isoforms are predominant in nervous and adipose tissues. The levels of expression of the different subunits vary according to cell and tissue type.
Suggested Companion Products
Preparation and Storage
Store undiluted at -20°C.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/pharmingen/protocols 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.
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.
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