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Our Abpromise guarantee covers the use of ab30682 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||1/100 - 1/150.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|WB||1/200 - 1/1000. Detects a band of approximately 126 kDa (predicted molecular weight: 126 kDa).Can be blocked with SREBP2 peptide (ab174740).
Block with 5% non-fat dry milk in TBS pH 7.4 for 1-2 hours at room temperature or over night at 4°C.
Blocking: 7% milk for 60 minutes at 20oC.
Immunohistochemistry (Frozen sections) analysis of wild type and ISR2 transgenic mouse jejunum tissue sections labelling SREBP2 with ab30682 at a dilution of 1/100. Sections (5–10 µm thickness) were cut from snap-frozen tissues embedded in OCT medium using a cryostat and were mounted on the slides and preserved at -80°C. Sections were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature followed by blocking in PBS containing 5% normal goat serum at room temperature. Sections were then incubated with the primary antibody in the blocking buffer. After PBS washes, the sections were incubated with the secondary antibody, an Alexa Fluor® 568-conjugated goat anti-rabbit IgG (red) for 60 min and then washed and mounted with slow-fade DAPI (blue, nuclei) by using coverslips. Microscopy was performed with a 20× oil immersion objective of Zeiss immunofluorescence microscope (Observer Z1) equipped with deconvolution software (AxioVision).
The figure shows predominant cytoplasmic staining in wild type mice and increased colocalization of SREBP2 with the nuclei in ISR2 mice (white arrow).
ICC/IF image of ab30682 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab30682, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
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