C7915-05C was used to detect phosphorylated CREB by immunoblot. 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. Immunoblot of indicated amounts 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 RT for 45 min. Blots were again rinsed 6X with TBST and then processed using ECL reagent (Amersham).
Immunohistochemistry. C7915-05C was used at 20 ug/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
C7915-05C was used to detect phosphorylated CREB by immunoblot. 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. Immunoblot of control (-) and in-vitro phosphorylated CREB (+) was used to show that the antibody reacts specifically with the phosphorylated form. Pan reactive CREB reacts equally with both non-phosphorylated and phosphorylated CREB (not shown). Detection occurs using a 1:500 dilution of antibody followed by 1:5,000 dilution of HRP Goat-a-Rabbit IgG with visualization via ECL. Film exposure was approximately 1’. Other detection systems will yield similar results. Personal Communication, Boss, J., Emory University School of Medicine, Atlanta, GA.
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