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+ TCF-4 set includes the TCF-4 antibody, a negative control normal mouse IgG, and qPCR primers which amplify a 198 bp region of human SP5 promoter. The TCF-4 and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of TCF-4-associated chromatin.
TCF4, also known as TCF7L2, is expressed widely during development. Gene targeting study indicates that it is required to maintain the crypt stem cells of the small intestine. TCF4 has many different splicing isoforms and they are expressed differentially in tissues and in cancers of different stages. Studies also indicate that variant of the TCF4 gene confers an increased risk of type 2 diabetes.
~66 kDa
Immunogen
Amino acids 31-331 of human TCF-4.
Application
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HT29 cells (3 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of Normal Mouse IgG or 2 µg Anti-TCF-4 and the Magna ChIP® G Kit (Cat. # 17-611).
Successful immunoprecipitation of TCF-4 associated DNA fragments was verified by qPCR using ChIP Primers, SP5 Promotor as a positive locus, and GAPDH promoter primers as a negative locus (Please see figures). 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 G (Cat. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
Representative lot data.
Cos-1 cells were transfected with TCF-4 cDNA in pUSE (lane 2) or pUSE (lane 1). RIPA lysates were prepared, resolved by electrophoresis, transferred to nitrocellulose and probed with anti-TCF-4 (1 mg/mL).
Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates TCF-4 (~66 kDa) (Please see figures).
Immunocytochemistry/Immunohistochemistry:
A previous lot of this antibody is reported to show positive immunostaining for TCF-4 in cells fixed with 4% paraformaldehyde/PBS (Amir, A. L., 2003).
Immunoprecipitation:
A previous lot of this antibody was reported to immunoprecipitate TCF-4 from SW620 whole cell lysate as a complex with Beta-Catenin (Persad, S., 2001).
Supershift Assay (EMSA):
A previous lot of this antibody was reported to supershift TCF-4/DNA complexes in electrophoretic mobility shift assays (EMSA) (Amir, A. L., 2003).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Transcription Factors
This ChIPAb+ TCF-4 -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
This antibody recognizes TCF-4
Packaging
25 assays per set. Recommended use: ~2 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Anti-TCF-4 (mouse monoclonal). One vial containing 50 µg of purified mouse monoclonal IgG2a in 50 µL of buffer containing 0.1 M Tris-glycine, pH 7.4, 0.15 M NaCl, 0.05% sodium azide and 30% glycerol. Store at -20°C.
Normal Mouse IgG. Two vials containing 25 µg of purified IgG in 25 µL of storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers, SP5 promote. One vial containing 75 μL of 5 μM of each primer specific for human SP5 promotor. Store at -20°C.
FOR: GGG TCT CCA GGC GGC AAG
REV: AGC GAA AGC AAA TCC TTT GAA TCC
Format: Purified
Protein G Chromatography
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: Variabillity in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HT29 cells (3 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of Normal Mouse IgG or 2 µg of Anti-TCF-4 and the Magna ChIP® G Kit (Cat. # 17-611).
Successful immunoprecipitation of TCF-4 associated DNA fragments was verified by qPCR using ChIP Primers, SP5 Promotor (Please see figures).
Please refer to the EZ-Magna ChIP G (Cat. # 17-409) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Control
Includes negative control normal mouse IgG and primers specific for human SP5 promoter.
Legal Information
GenBank is a registered trademark of United States Department of Health and Human Services
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.
