Description
cAMP-dependent Protein Kinase (PKA) is composed of two distinct subunits: catalytic (C) and regulatory (R). Four regulatory subunits have been identified: RIα, RIβ, RIIα, and RIIβ. These subunits define type I and II PKAs. 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, Cβ, or Cγ). Most cells, including T lymphocytes, express both type I and type II PKAs. RIIα expression is associated with cellular transformation, while RIIβ expression correlates with mitotic arrest and cellular differentiation. Type II PKA can be distinguished by autophosphorylation of the R subunits, while type I PKA binds Mg/ATP with high affinity. The cAMP-dependent autophosphorylation of the human RIIβ subunits occurs at serine 114 (S114). In addition to their enzyme regulatory activity, the RIIα and RIIβ subunits determine the subcellular location of the holoenzymes via their interactions with specific intracellular anchoring proteins. The 47/PKA monoclonal antibody recognizes the phosphorylated S114 in the RIIβ subunit of PKA. The orthologous phosphorylation site in mouse and rat PKA[RIIβ] is S112.
Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation. Any of the three BD Phosflow™ permeabilization buffers may be used.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
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. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
8. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
