711615

antibody from Invitrogen Antibodies

Targeting: PRKACB PKACb

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711615

Provider

Invitrogen Antibodies

Product name

Phospho-PKA alpha/beta (Thr197) Recombinant Polyclonal Antibody (24HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

24HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (1), HeLa treated with CalyculinA (Serum starved for 24hrs, 0.1 µg/mL for 30mins) (2). The blots were probed with Anti-PKA CAT alpha/beta (pT197) Recombinant Rabbit Polyclonal Antibody (Product # 711615, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 40 kDa band corresponding to PKA CAT alpha/beta (pT197) was observed. To confirm the specificity of Anti-PKA CAT alpha/beta (pT197) Recombinant Rabbit Polyclonal Antibody, competition was performed with the phosphopeptide (10 µg/mL) as shown in the second blot and non-phosphopeptide (10 µg/mL) as shown in the third blot. The phosphopeptide competes with the antibody and prevents it from binding to the target protein. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of PKA CAT ALPHA/BETA (pT197) using Anti-PKA CAT ALPHA/BETA (pT197) Recombinant Rabbit Polyclonal Antibody (Product # 711615, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of PKA CAT ALPHA/BETA (pT197) protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of PKA CAT ALPHA/BETA (pT197) in cells treated with calyculin A (100 ng/mL, 30 min). Panel e) shows reduced signal in untreated cells. Panel f) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.