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Purified Mouse Anti-PKA RIIβ (pS114)
Purified Mouse Anti-PKA RIIβ (pS114)

Left: Westen Blot: Rat Cerebrum lysate was either left untreated (lane 1) or treated (lane 2) with 150U/ml of lambda phosphatase for 1 hour at 37°C. The top panel was probed with PKA RIIβ (cat. no. 610625). The bottom panel was probed with anti-PKA RIIβ (pS114). Recommended to use antibody in range of 1:1000 dilution.

Right: Immunofluorescent staining of SK-N-SH cells.  Cells were seeded in a 384 well collagen coated Microplates (Material # 353962) at ~ 8,000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the anti- PKARIIb p S114 antibody.  The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen).  The image was taken on a Pathway 855 or 435 imager using a 20x objective.  This antibody also stained SH-SY5Y, and C6 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure).

Left: Westen Blot: Rat Cerebrum lysate was either left untreated (lane 1) or treated (lane 2) with 150U/ml of lambda phosphatase for 1 hour at 37°C. The top panel was probed with PKA RIIβ (cat. no. 610625). The bottom panel was probed with anti-PKA RIIβ (pS114). Recommended to use antibody in range of 1:1000 dilution.

Right: Immunofluorescent staining of SK-N-SH cells.  Cells were seeded in a 384 well collagen coated Microplates (Material # 353962) at ~ 8,000 cells per well.  After overnight incubation, cells were stained using the methanol fix/perm protocol (see Recommended Assay Procedure) and the anti- PKARIIb p S114 antibody.  The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen).  The image was taken on a Pathway 855 or 435 imager using a 20x objective.  This antibody also stained SH-SY5Y, and C6 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Rat (QC Testing), Human, Mouse (Tested in Development)
Mouse IgG1, κ
Phosphorylated Human PKA[RIIβ] peptide Peptide
Western blot (Routinely Tested), Flow cytometry, Fluorescence microscopy (Tested During Development)
53 kDa
250 µg/ml
AB_399845
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at -20°C.

Recommended Assay Procedures

Methanol Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 90% methanol. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Wash three times with PBS. Flick out PBS and add second step reagent. Incubate for 1 hour at RT. Wash three times with PBS. Image sample.

Triton-X 100 Procedure for a 96 well plate:

Remove media from wells. Add 100 µl/well fresh 3.7% Formaldehyde in PBS. Incubate for 10 minutes at room temperature (RT). Flick out and add 100 µl/well 0.1% Triton-X 100. Incubate for 5 minutes at RT. Flick out and wash twice with PBS. Flick out PBS and add 100 µl/well blocking buffer (3% FBS in PBS). Incubate for 30 minutes at RT. Flick out and add diluted antibody (diluted in blocking buffer). Incubate for 1 hour at RT. Flick out and wash three times with PBS. Flick out and add second step reagent. Incubate for 1 hour at RT. Flick out and wash three times with PBS. Image sample.

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. 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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
612550 Rev. 1
Antibody Details
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47/PKA

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.

612550 Rev. 1
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
612550 Rev.1
Citations & References
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Development References (2)

  1. Budillon A, Cereseto A, Kondrashin A, et al. Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts. Proc Natl Acad Sci U S A. 1995; 92(23):10634-10638. (Biology). View Reference
  2. Keryer G, Luo Z, Cavadore JC, Erlichman J, Bornens M. Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2. Proc Natl Acad Sci U S A. 1993; 90(12):5418-5422. (Biology). View Reference
612550 Rev. 1

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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.