Figure 2. The relationship between the protein concentration of lysates from untreated and forskolin-treated SK-N-MC cells and the absorbance at 450 nm using the PathScan® Phospho-CREB (Ser133) Sandwich ELISA Kit is shown.
Figure 1. Treatment of SK-N-MC cells with forskolin stimulates phosphorylation of CREB at Ser133, detected by the PathScan® Phospho-CREB (Ser133) Sandwich ELISA Kit #7385, but does not affect the levels of total CREB detected by PathScan® Total CREB Sandwich ELISA Kit #7390. SK-N-MC cells (80-90% confluent) were treated with 120 μM forskolin with 0.5 mM IBMX for 30 minutes and lysed. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using CREB (48H2) Rabbit mAb #9197 (left panel) or Phospho-CREB (Ser133) (1B6) Mouse mAb #9196 (right panel) are shown in the bottom figure.
|REACTIVITY||H M R|
|Product Includes||Volume||Solution Color|
|CREB Rabbit mAb Coated Microwells||96 tests|
|Phospho-CREB (Ser133) Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml|
The PathScan® Phospho-CREB (Ser133) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of CREB when phosphorylated at Ser133. A CREB rabbit antibody has been coated onto the microwells. After incubation with cell lysates, CREB protein (phosphorylated and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-CREB (Ser133) mouse monoclonal detection antibody is added to detect the captured phospho-CREB (Ser133) protein. HRP-linked anti-mouse IgG is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of CREB phosphorylated at Ser133.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
revised January 2016
Protocol Id: 205
PathScan® Phospho-CREB (Ser133) Sandwich ELISA Kit #7385 detects endogenous levels of CREB protein when phosphorylated at Ser133 as shown in Figure 1. Kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. This kit also detects the phosphorylated form of the CREB-related protein ATF-1.
Human, Mouse, Rat
CREB is a bZIP transcription factor that activates target genes through cAMP response elements. CREB is able to mediate signals from numerous physiological stimuli, resulting in regulation of a broad array of cellular responses. While CREB is expressed in numerous tissues, it plays a large regulatory role in the nervous system. CREB is believed to play a key role in promoting neuronal survival, precursor proliferation, neurite outgrowth, and neuronal differentiation in certain neuronal populations (1-3). Additionally, CREB signaling is involved in learning and memory in several organisms (4-6). CREB is able to selectively activate numerous downstream genes through interactions with different dimerization partners. CREB is activated by phosphorylation at Ser133 by various signaling pathways including Erk, Ca2+, and stress signaling. Some of the kinases involved in phosphorylating CREB at Ser133 are p90RSK, MSK, CaMKIV, and MAPKAPK-2 (7-9).
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