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FITC Mouse Anti-PKA [RI]
FITC Mouse Anti-PKA [RI]

Immunofluorescent staining of HeLa cells.  HeLa cells (Human cervical epitheloid carcinoma; ATCC CCL-2.2) were seeded in a BD Falcon™ 96-well imaging plate (Cat. No. 353219) at ~ 10,000 cells per well.  After overnight incubation, cells were stained using the Triton-X 100 fix/perm protocol (see Recommended Assay Procedure) and the FITC mouse anti-PKA [RI] antibody.  The image was taken on a Pathway 855 imager using a 20x objective.  This antibody also stained A549 and U2OS cells using either the Triton-X 100 or Methanol fix/perm protocols (see Recommended Assay Procedure).

Immunofluorescent staining of HeLa cells.  HeLa cells (Human cervical epitheloid carcinoma; ATCC CCL-2.2) were seeded in a BD Falcon™ 96-well imaging plate (Cat. No. 353219) at ~ 10,000 cells per well.  After overnight incubation, cells were stained using the Triton-X 100 fix/perm protocol (see Recommended Assay Procedure) and the FITC mouse anti-PKA [RI] antibody.  The image was taken on a Pathway 855 imager using a 20x objective.  This antibody also stained A549 and U2OS cells using either the Triton-X 100 or Methanol fix/perm protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog, Chicken, Frog (Tested in Development)
Mouse IgG2b
Mouse PKA [RI] subunit aa. 225-381
Bioimaging (Routinely Tested), Immunofluorescence (Tested During Development)
48 kDa
250 µg/ml
AB_397568
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. The antibody was conjugated with FITC under optimum conditions, and unreacted FITC was removed. Store undiluted at -20°C.

Recommended Assay Procedures

Bioimaging:  Please refer to http://www.bdbiosciences.com/pharmingen/protocols/Bioimaging_Certified.shtml

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.
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Antibody Details
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18/PKA [RI]

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.

610167 Rev. 1
Format Details
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FITC
Fluorescein (FITC) is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 518-nm. FITC is designed to be excited by the Blue laser (488-nm) and detected using an optical filter centered near 520 nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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FITC
Blue 488 nm
494 nm
518 nm
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Citations & References
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Development References (4)

  1. Cho-Chung YS. Role of cyclic AMP receptor proteins in growth, differentiation, and suppression of malignancy: new approaches to therapy. Cancer Res. 1990; 50(22):7093-7100. (Biology). View Reference
  2. Dohrman DP, Diamond I, Gordon AS. Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus. Proc Natl Acad Sci U S A. 1996; 93(19):10217-10221. (Biology). View Reference
  3. Rohlff C, Clair T, Cho-Chung YS. 8-Cl-cAMP induces truncation and down-regulation of the RI alpha subunit and up-regulation of the RII beta subunit of cAMP-dependent protein kinase leading to type II holoenzyme-dependent growth inhibition and differentiation of HL-60 leukemia cells. J Biol Chem. 1993; 268(8):5774-5782. (Biology). View Reference
  4. Taylor SS, Buechler JA, Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem. 1990; 59:971-1005. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.