Immunocytochemistry/ Immunofluorescence - SREBP1 antibody [2A4] (ab3259)

ICC/IF image of ab3259 stained HeLa cells. The cells were 4% formaldehyde 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 (ab3259, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.

Western blot - SREBP1 antibody [2A4] (ab3259)

Anti-SREBP1 antibody [2A4] (ab3259) at 1 µg/ml + HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate (ab52257) at 10 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 127 kDa
Observed band size : 127 kDa
Additional bands at : 36 kDa,65 kDa (possible cleavage fragment). We are unsure as to the identity of these extra bands.

Exposure time : 12 minutes

The band observed at 65 kDa is believed to be the cleaved mature form of SREBP1.

Flow Cytometry-SREBP1 antibody [2A4](ab3259)

Overlay histogram showing THP1 cells stained with ab3259 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab3259, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in THP1 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Immunocytochemistry/ Immunofluorescence - SREBP1 antibody [2A4] (ab3259)

ICC/IF image of ab3259 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 (ab3259, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-mouse IgG - H&L, pre-adsorbed (ab96879) used at a 1/250 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.

Western blot - Anti-SREBP1 antibody [2A4] (ab3259)

Anti-SREBP1 antibody [2A4] (ab3259) at 1/1000 dilution + whole cell lysate prepared from murine macrophages at 30 µg

Secondary
HRP conjugated goat anti-mouse IgG polyclonal at 1/2000 dilution
developed using the ECL technique

Predicted band size : 127 kDa


Exposure time : 3 minutes

The two bands have the expected size corresponding to the full and cleaved forms of SREBP1.

Image courtesy of an anonymous Abreview.