Chromatin immunoprecipitation analysis of p300 in mouse kidney was performed using biotinylated p300 antibody (Product # 730044). Kidney preparation: CD1 mice (Charles River Laboratories, Wilmington, MA; 6-10 weeks of age, male; 30-35 gms), received a tail vein injection of either LPS (2 mg/kg; 0111:B4; in 80 µl of PBS) or PBS. Two hours after injection, mice were anesthetized, the abdominal cavity was opened, the kidneys were extracted, and renal cortical samples were cut from the kidneys with a razor blade and rapidly frozen at -70oC. The mice died within 30 sec due to exsanguinations. Chromatin preparation and multiplex Matrix ChIP platform: The multiplex microplate Matrix ChIP method was previously described (see reference for Matrix ChIP protocol: (http://www.ncbi.nlm.nih.gov/pubmed/22098709 ). Briefly, kidney fragments (10-20 mg) were cross-linked with formaldehyde, and chromatin was sheared. ChIP assays were done using either protein A- or streptavidin-coated 96-well polypropylene microplates. p300-biotin antibody (Product # 730044) was used at 0.5 ug/well. For Pol II antibody (0.5ug/well) testing, wells were pre-incubated with 1ug/well of goat anti-mouse biotinylated antibody (Product # B2763). 1ul of eluted DNA was used in 2 ul real-time PCR reactions (ABI7900HT) reactions containing primers shown to amplify exon 1 and exon 7 of the ICAM1 gene. PCR calibration curves were generated for each primer pair from a dilution series of total mouse or human genomic D
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Human|
|Host / Isotype||Mouse / IgG1|
|Immunogen||1038-1161 of 2414 of EP300 Protein.|
|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 *|
|ChIP assay (ChIP)||1:100|
|Immunoprecipitation (IP)||Assay Dependent|
* 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.
|Immunoprecipitation (IP)||See 1 publications below|
730044 has been successfully used in ChIP analysis of p300 in mouse kidney.
EP300 encodes the adenovirus E1A-associated cellular p300 transcriptional co-activator protein. p300 is related by sequence to CPB (CREB-binding protein [CREB: cyclic-AMP responsive element binding protein]), and like CPB can stimulate transcription through activation of CREB. This EP300 activity is specifically inhibited by the adenovirus oncoprotein E1A. EP300 has also been identified as a co-activator of HIF1A (hypoxia-inducible factor 1 alpha), and thus plays a role in the stimulation of hypoxia-induced genes such as VEGF.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.
730044 was used in immunoprecipitation to describe a mass spectrometry-based method for scoring immunoprecipitation antibody quality
|Marcon E,Jain H,Bhattacharya A,Guo H,Phanse S,Pu S,Byram G,Collins BC,Dowdell E,Fenner M,Guo X,Hutchinson A,Kennedy JJ,Krastins B,Larsen B,Lin ZY,Lopez MF,Loppnau P,Miersch S,Nguyen T,Olsen JB,Paduch M,Ravichandran M,Seitova A,Vadali G,Vogelsang MS,Whiteaker JR,Zhong G,Zhong N,Zhao L,Aebersold R,Arrowsmith CH,Emili A,Frappier L,Gingras AC,Gstaiger M,Paulovich AG,Koide S,Kossiakoff AA,Sidhu SS,Wodak SJ,Gräslund S,Greenblatt JF,Edwards AM||Nature methods (12:725)||2015|