Description
General description
All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
The ChIPAb+ Phospho-CREB (Ser133) set includes the Phospho-CREB (Ser133) antibody, a negative control normal rabbit IgG, and qPCR primers which amplify a 64 bp region of human cFos CRE. The Phospho-CREB (Ser133) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Phospho-CREB (Ser133)-associated chromatin.
CREB is a β-ZIP 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. CREB promotes outgrowth and differentiation as a mediator of neurotrophin pathways. 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.
Detects CREB only when phosphorylated on Ser133 (~43 kDa). May recognize ATF-1 (~38 kDa) and CREM (~30 kDa) as the two proteins share significant homology to the phosphorylated immunizing peptide.
Immunogen
Epitope: phospho-Ser133
KLH-conjugated synthetic peptide corresponding to the amino acids including and encompassing phosphorylated Ser133 of CREB.
Application
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from 293T cells (5 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 4 µg of Normal Rabbit IgG, (Part No. P64B), or 4 µg of Anti-Phospho-CREB (Ser133) antibody (Part No. CS204400) and the Magna ChIP® A Kit (Cat. # 17-610).
Successful immunoprecipitation of Phospho-CREB (Ser133) antibody associated DNA fragments was verified by qPCR using ChIP Primers cFos CRE (Part No. CS203203) as a positive locus, and cFos downstream 4Kb as a negative locus (Figure 2). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
Representative lot data.
Untreated (lane 1) and Forskolin-treated (Lane 2) NIH/3T3 lysates were resolved by SDS-PAGE, transferred to PVDF, and probed with anti-phospho-CREB (Ser133) (1:1,000 dilution).
Proteins were visualized using a donkey anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates phosphorylated CREB (Figure 3).
Immunoprecipitation:
10 µL from a representative lot of this antibody was shown to immunoprecipitate phosphorylated CREB from 500 µg of cells treated with 50 mM forskolin.
Immunohistochemistry: A 1:1000 dilution of a representative lot detected phosphorylated CREB in formalin-fixed paraffin-embedded normal and ischemic rat brain sections.
Electrophoretic Mobility Shift:
A representative lot of this antibody has been shown to gel shift by an independent laboratory.
Research Category
Signaling
Epigenetics & Nuclear Function
Research Sub Category
Transcription Factors
This ChIPAb+ Phospho-CREB (Ser133) -ChIP Validated Antibody & Primer Set (Antibody ChIP) conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
Recognizes p43 phosphorylated CREB. May also recognize p30 and p38 kDa proteins. The p30 & p38 proteins may be phosphorylated CREM and ATF-1 respectively, since the two proteins share significant homology to the phosphorylated CREB immunizing peptide.
Packaging
25 assays per set. Recommended use: ~4 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-phospho-CREB (Ser133) (rabbit polyclonal), Part No. CS204400. One vial containing 100 µg of affinity purified rabbit serum in 0.1 M Tris-Glycine (pH 7.4) 0.15 M NaCl, and 0.05% sodium azide, before the addition of 30% glycerol. Store at -20°C.
Normal Rabbit IgG, Part No. PP64B. One vial containing 125 µg Rabbit IgG in 125 µL storage buffer containing 0.05% sodium azide.
Store at -20°C.
ChIP Primers cFos CRE, Part No. CS203203. One vial containing 75 μL of 5 μM of each primer specific for the cFos CRE. Store at -20°C.
FOR: GGC CCA CGA GAC CTC TGA GAC A
REV: GCC TTG GCG CGT GTC CTA ATC T
Format: Purified
Purified by protein A-Sepharose followed by immunoadsorbant chromatography using an unphosphorylated CREB affinity gel column.
Preparation Note
Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance. Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from 293T cells (5 X 10E6 cell equivalents per IP) was subjected to chromatin immunoprecipitation using either 4 μg of a Normal Rabbit IgG , or 4 μg of Anti-Phospho-CREB antibody and the Magna ChIP® A Kit (Cat. # 17-610). Successful immunoprecipitation of Phospho-CREB (Ser133) associated DNA fragments was verified by qPCR using ChIP Primers cFos CRE (Figure 1).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Control
Includes negative control normal rabbit IgG and primers specific for human cFOS CRE.
Other Notes
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
Legal Information
MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany
UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
