Western blot analysis of CREB (pS133) was performed by loading 20 µg of MDA-MB-231 (lane1), MCF7 (lane2), A549 (lane3), A431 (lane4) and HEK-293 (lane5) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (NP0321BOX), XCell SureLock™ Electrophoresis System (EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. CREB (ps133) was detected at ~43 kDa using ABfinity™ CREB Monoclonal Antibody (700129) at 1-3 µg/ml in 2.5 % skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with phosphopeptide (10 µg/mL) as shown in the corresponding blot on right. Goat Anti-Rabbit IgG-HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1-3µg/10^6 cells|
|Western Blot (WB)||1-3µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
CREB (cAMP responsive element binding protein) encodes for a 43 kDa protein transcription factor and belongs to leucine zipper family. CREB binds to the cyclic-AMP response element (CRE, a sequence present in many viral and cellular promoters) as a homodimer. Phosphorylated form of CREB act as an inducer of transcription for the genes, upon hormone dependent stimulation of cyclic-AMP pathway. CREB is known to play important role in circadian rythm generation and memory formation; aberration in CREB-mediated gene expression has been linked with Alzheimer disease. In humans, the gene is located on the q arm of chromosome 2.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.