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+ CREB, antibody/primer set includes the CREB, antibody, a negative control antibody (purified rabbit IgG), and qPCR primers which amplify a 64 bp region flanking the CRE of the human cFos gene. The CREB and negative control cultured supernatant are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of CREB associated chromatin.
Every lot of the ChIPAb+ line of antibodies is individually validated for chromatin precipitation, in order to guarantee successful ChIP assays every time. Each antibody includes a control primer set for performance confirmation. CREB antibody and the negative control antibody (rabbit normal IgG) can be used to demonstrate that the CREB antibody is functionally validated in the precipitation of CREB associated chromatin.
The qPCR primers included flank the CREB binding site in human NR4A2 promoter.
~43 kDa
Immunogen
CREB purified antibody is made against a KLH-conjugated synthetic peptide corre-sponding to amino acids 5-24 of human CREB.
Epitope: a.a. 5-24
Application
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from 293T cells (5 X 10⁶ cell equivalents per IP) was subjected to chromatin immunoprecipitation using 10 µL of either a negative supernatant, or CREB antibody and the Magna ChIP® A Kit (Cat. # 17-610).
Successful immunoprecipitation of CREB-associated DNA fragments was verified by qPCR using cFos downstream 4Kb (negative) and cFos CRE (positive) (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 A ChIP (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
Representative lot data.
HeLa nuclear extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-CREB, clone NL904 (1:8,000 dilution)
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates CREB (~43 kDa).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Chromatin Biology
This ChIPAb+ CREB -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
Not tested in other species. The immunogen sequence is identical in a wide range of animal and plant species, so broad cross-reactivity is expected.
Recognizes CREB, MW ~43 kDa.
Packaging
25 assays per set, ~10 μL per chromatin immunoprecipitation.
Physical form
25 assays per set. Recommended use: ~10 μL antibody per chromatin immunoprecipitation (dependent upon biological context).
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.
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from 293T cells (5 X 10⁶ cell equivalents per IP) were subjected to chromatin immuno-precipitation using 10 µL of either a negative control cultured supernatant, or CREB antibody and the Magna ChIP® A Kit (Cat. # 17-610). Successful immunoprecipitation of CREB-associated DNA fragments was verified by qPCR using Control Primers (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 cultured supernatant and primers specific for of human cFos CRE.
Other Notes
Anti-CREB (Rabbit monoclonal IgG): Part No. CS203204. One vial containing 250 μL of cultured supernatant containing 0.05% sodium azide. Store at -20°C.
Negative Control Supernatant: Part No. CS203202. One vial containing 250 μL of cultured supernatant 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
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.
