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Our customers have provided mixed feedback about the Chip application. The data suggests some lots worked perfect but some completely failed. We will be happy to answer any question from potential users. Customers can contact scientific support for latest information about the lot specific data.
Our Abpromise guarantee covers the use of ab5089 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 5 - 10 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. PubMed: 20018680|
|ChIP||Use at an assay dependent concentration.|
|WB||Use a concentration of 0.3 - 1 µg/ml. Detects a band of approximately 50 kDa (predicted molecular weight: 49.1 kDa).Can be blocked with Human FOXA1 peptide (ab23074).
An additional band of unknown identity was also consistently observed at 37kDa. This band was successfully blocked by incubation with the immunizing peptide.
Goat polyclonal to FOXA1 antibody (ab5089) was used in a ChIP assay to detect in vivo binding of FOXA1/HNF3a to the promoter of the TFF1 gene in MCF-7 cells. The cells were fixed in 1% formaldehyde for 15 minutes at room temperature and ChIP was performed using 6µg/ml ab5089. Real-time PCR was used to quantify the enrichment of the TFF1 promoter in the FOXA1 ChIP'd DNA, using the input DNA as the reference. The results indicate that there is an 18-fold enrichment of the TFF1 promoter in the DNA ChIp'd with ab5089.
This image was submitted as part of a review.
Chromatin was prepared from nuclear lysate of the human MCF7 breast epithelial adenocarcinoma cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in formaldehyde. The primary antibody was diluted to 0.2 μg/μg chromatin and incubated with the sample for 16 hours at 4°C in a dilution buffer that contains SDS, DOC, Triton X-100, EDTA, HEPES, NaCl. The immunoprecipitated DNA was quantified by real time PCR. Ct values were converted to DNA copy numbers using a standard curve in the Q-PCR step. The number of binding events detected for each test reaction was then calculated by taking into account the DNA copy number, cell equivalents of chromatin used in the ChIP and PCR, and primer pair amplification efficiency.
An additional band of unknown identity was also consistently observed at 37kDa.