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Our Abpromise guarantee covers the use of ab2923 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|EMSA||Use at an assay dependent concentration. PubMed: 18429957|
|EMSA||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.
|WB||1/1000 - 1/10000. Detects a band of approximately 83 kDa (predicted molecular weight: 57 kDa).|
|ELISA||Use at an assay dependent concentration.|
|ChIP||Use at an assay dependent concentration.|
Immunocytochemistry/Immunofluorescence analysis of HSF1 (green) in HeLa and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were probed with ab2923 (1:50) for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat-anti rabbit IgG secondary antibody (1:400) for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.
Immunoprecipitation of HSF1 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of ab2923 overnight on a rocking platform at 4°C. Immune complexes were captured on 50µl Protein A/G Plus Agarose, washed extensively, and eluted with buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was incubated with ab2923 (1:1000) overnight rotating at 4°C, washed in TBST, and probed with detection reagent (1:1000) for at least one hour. Chemiluminescent detection was performed.
Western blot analysis of Heat Shock Factor 1 (HSF1) was performed by loading 50µg of the indicated whole cell lysates per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was incubated with ab2923 (1/1000) overnight at 4°C on a rocking platform, washed in TBS-0.1% Tween 20, and probed with a HRP-conjugated goat anti-rabbit IgG secondary antibody (1/20,000) for at least one hour. Chemiluminescent detection was performed.
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