Analysis Note

ControlIncludes negative control mouse ascites and primers specific for human IκBα promoter.

Application

Research Sub CategoryRNA Metabolism & Binding Proteins

The ChIPAb+ SMRT set includes the SMRT antibody, a negative control mouse ascites & qPCR primers which amplify a 299 bp region of human IκBα promoter.

Research CategoryEpigenetics & Nuclear Function

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from serum starved, TNFα treated (20 ng/mL, 30 min) HeLa cells (~3 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of Negative Ascites or 2 µL Anti-SMRT) and the Magna ChIP™ G Kit (Cat. # 17-611).
Successful immunoprecipitation of SMRT associated DNA fragments was verified by qPCR using ChIP Primers, IκBα promoter as a positive locus, and β-Actin 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™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.

Western Blot Analysis:
Representative lot data.
Human brain lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with Anti-SMRT (1:1000 dilution).
Proteins were visualized using a Goat Anti-Mouse IgG conjugated to HRP and a chemiluminescence detection system (Please see figures).

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.

General description

The Silencing mediator of retinoic acid & thyroid hormone receptor protein, commonly called the SMRT protein mediates the transcriptional repression of some nuclear receptors by promoting chromatin condensation, thus preventing access of the basal transcription machinery. Consistent with this activity, this protein is known to form a large corepressor complex containing SIN3A/B and histone deacetylases HDAC1 and HDAC2. SMRT is also a component of the N-Cor repressor (nuclear receptor corepressor), a multi-subunit complex minimally composed of NCOR1, NCOR2, HDAC3, TBL1X, TBL1R, CORO2A and GPS2. SMRT and nuclear receptor corepressor N-CoR are related transcriptional corepressors which contain two distinct domains capable of interacting with unliganded nuclear receptors to repress their basal transcriptional activity.

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+ SMRT set includes the SMRT antibody, a negative control mouse ascites, and qPCR primers which amplify a 299 bp region of human IκBα promoter. The SMRT and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of SMRT-associated chromatin.

Immunogen

Recombinant protein corresponding to human SMRT.

Legal Information

MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Packaging

25 assays per set. Recommended use: ~2 µL of antibody per chromatin immunoprecipitation (dependent upon biological context).

Physical form

Anti-SMRT (mouse monoclonal). One vial containing 50 µL of unpurified mouse monoclonal ascites with 0.05% sodium azide. Store at -20°C.

Negative Ascites (mouse). One vial containing 50 µL of mouse ascites with 0.05% sodium azide. Store at -20°C.

ChIP Primers, IκBα promoter. One vial containing 75 µL of 5 µM of each primer specific for human IκBα promoter. Store at -20°C.
FOR: GAC GAC CCC AAT TCA AAT CG
REV: TCA GGC TCG GGG AAT TTC C

Unpurified

Quality

Chromatin Immunoprecipitation:
Sonicated chromatin prepared from serum starved, TNFα treated (20 ng/mL, 30 min) HeLa cells (~3 X 10⁶ cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of Negative Ascites or 2 µL Anti-SMRT) and the Magna ChIP^® G Kit (Cat. # 17-611).
Successful immunoprecipitation of SMRT associated DNA fragments was verified by qPCR using ChIP Primers, IκBα promoter (Please see figures).

Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.

Specificity

Recognizes SMRT.

Storage and Stability

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.

Target description

~ 275 kDa