The assay is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standard dilutions and samples are added to the wells precoated with capture antibody specific for CREB and incubated 2 hours at RT. CREB antigen binds to the capture antibody. A detection antibody specific for either non-phosphorylated or phosphorylated protein is incubated 1 hr at RT. An anti-rabbit IgG-HRP binds to the immobilized protein completing the four-member sandwich. The reaction is visualized by TMB substrate, followed by the stop solution. The intensity of the yellow color, measured in a multiwell plate reader at 450 nm, is directly proportional to the concentration of CREB in original sample. The unknown concentrations are calculated from the standard curve run with each assay.
Detects and quantifies CREB phosphorylated on serine 133 in lysates of human and mouse cells. For normalizing the phospho-CREB (pSer133) content of the samples, a CREB ELISA independent of phosphorylation status is available from Sigma Product No. CS0580.
CREB, also referred to as cAMP-Response Element-Binding protein, activates transcription in response to stimuli that elevate cytoplasmic cAMP concentrations.
Features and Benefits
CREB ELISA is a fast (total time 4 hours), sensitive, and specific alternative to immunoblotting and RIA. The ease of use is facilitated by precoated plates and ready-to-use reagents.
The analytical sensitivity is <0.09 units/mL. The specificity for phosphorylated CREB (pSer133) was confirmed by peptide competition.