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Purified wheat germ RNA polymerase II.
Our Abpromise guarantee covers the use of ab817 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|CHIPseq||Use at an assay dependent concentration. PubMed: 19251593Use 2ug per 0.3ml of sonicated chromatin.|
|ICC||Use at an assay dependent concentration.|
|Purification||Use at an assay dependent concentration.|
|WB||Use at an assay dependent concentration. Detects a band of approximately 217 kDa (predicted molecular weight: 217 kDa).|
|ICC/IF||Use at an assay dependent concentration.|
|IHC - Wholemount||Use at an assay dependent concentration.
|ChIP||Use at an assay dependent concentration. See Abreview; the user recommends using anti-mouse IgG coated Dynabeads instead of Protein A to recover the precipitate).|
|Flow Cyt||Use 0.5µg for 106 cells.|
|IP||Use at an assay dependent concentration.|
Various regions across the Actin2/7 loci were tested for the presence of RNA polymerase II CTD repeat YSPTSPS. A nuclear lysate from Arabidopsis thaliana seedlings was crosslinked using 1% formaldehyde for 30 seconds. The ChIP was performed with 0.1 µg of ab817 per µg of chromatin; incubated together for 16 hours at 4°C. The immunoprecipitated DNA was quantified by Real-Time PCR. The bottom panel indicates the positive (ab817) and negative controls (no antibody) at region B3.
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 used in concentration of 0.2 µg/µg chromatin and incubated with the sample for 16 hours at 4°C in SDS, DOC, TritonX-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.
Overlay histogram showing HeLa cells stained with ab817 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab817, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG, H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
IHC - Wholemount of Caenorhabditis elegans embryo labelling RNA polymerase II CTD repeat YSPTSPS with ab817. Sample was incubated with primary antibody (1/100 in PBS + 3% BSA + 0.1% Triton-X 100) for 24 hours at 4°C. ab150113, a goat anti-mouse Alexa Flour® 488 (undiluted) was used as the secondary antibody.
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