CREB phospho S133 Antibody

Anti-CREB pS133 was used to detect phosphorylated CREB by western blot at ~46kDa. Recombinant His-tagged human CREB was produced in E.coli and purified by metal affinity chromatography. An aliquot of purified CREB was phosphorylated in-vitro using Protein Kinase A and ATP. Western blot of control (-) and in-vitro phosphorylated CREB (+) was used to show that the antibody reacts specifically with the phosphorylated form. Pan reactive CREB (orb750462) reacts equally with both non-phosphorylated and phosphorylated CREB (not shown). Detection occurs using a 1:500 dilution of antibody followed by 1:5000 dilution of HRP Goat-a-Rabbit IgG with visualization via ECL. Film exposure was approximately 1'. Other detection systems will yield similar results.

Anti-CREB pS133 was used to detect phosphorylated CREB by western blot. Recombinant His-tagged human CREB was produced in E.coli and purified by metal affinity chromatography. An aliquot of purified CREB was phosphorylated in-vitro using Protein Kinase A and ATP. Western blot of indicated amounts (100 ng, 50 ng, 25 ng, 12.5 ng) of control (-) and in-vitro phosphorylated CREB (P) were loaded to show that the antibody reacts specifically with the phosphorylated form. Blots were blocked in 5% milk in TBS + 0.1% Tween-20 (TBST-M) overnight at 4°C. Detection occurs using a 1:500 dilution of antibody diluted in TBST-M and incubated at room temperature with rocking for 1 hour. Blots were rinsed 6X with TBST and incubated with goat anti-rabbit-HRP at 1:5000 in TBST-M at room temperature for 45 min. Blots were again rinsed 6X with TBST and then processed using ECL reagent (Amersham) according to manufacturer's instructions. Exposure time: 1 min using Kodak Biomax MR film. Screaton, The Salk Institute for Biological Studies.

Biorbyt's affinity purified anti-CREB pS133 antibody was used at 20 µg/ml to detect signal in a variety of tissues including multi-human, multi-brain and multi-cancer slides. This image shows moderate to strong nuclear staining of tonsillar lymphocytes. Tissue was formalin-fixed and paraffin embedded. The image shows localization of the antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain.