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Our Abpromise guarantee covers the use of ab53773 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/500 - 1/1000. Detects a band of approximately 100 kDa (predicted molecular weight: 69 kDa).|
|IHC-P||Use a concentration of 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ICC/IF||1/100 - 1/250.|
|ChIP||Use at an assay dependent concentration. PubMed: 24019952|
|IP||Use at an assay dependent concentration. PubMed: 23222847|
ab53773 (1/200) staining PML protein in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells (green). Cells were fixed with methanol, permeabilized with triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.
Chromatin was prepared from modified murine D3 ES cells (an in vitro model of RB1 imprinted expression was generated by integrating human PPP1R26P1 into intron 2 of mouse Rb1 in murine embryonic stem cells (mESCs)). Cells were fixed with formaldehyde for 10 minutes. Chromatin was sheared into fragment sizes ranging from 200 to 600 bp. For immunoprecipitation 50 μl of sheared chromatin was set aside as input and 20-40 μg of sheared chromatin in a volume of 100 μl was used for antibody incubation. 4 µg of ab53773 to PML was used as a non-related control (black bars) and 4 µl of a rabbit polyclonal to RNAPII CTD repeat YSPTSPS (phospho S5) (shaded bars) was added as the test. The immunoprecipitated DNA was quantified at 3 positions on the the retinoblastoma (Rb1) promoter. 1,2 = Rb1_48; 3,4 = Rb1_69 and 5,6 = Rb1_74
ab53773 staining PML in human normal colon tissue. Staining is localized to the nucleus.
Left panel: ab53773 at 4 µg/ml. Right panel: Isotype control.
Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved with the EDTA pH 9.0 buffer. Slides were blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with hematoxylin and coverslipped. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
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