Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: ERp57 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HepG2 whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab13506 observed at 57 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab13506 was shown to specifically react with ERp57 in wild-type HAP1 cells as signal was lost in ERp57 knockout cells. Wild-type and ERp57 knockout samples were subjected to SDS-PAGE. ab13506 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 0.5 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ICC/IF image of ab13506 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 ab13506 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) IgG (H+L) 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.

ERp57 antibody (ab13506) at 1/1000 dilution (in PBST) for 8 hours at 4°C + lysate of mouse of pancreatic cancer cell line at 25 µg.

Secondary
An HRP conjugated Sheep anti-mouse IgG polyclonal at 1/5000 dilution
developed using the ECL technique

Performed under non-reducing conditions.

Exposure time: 1 minute

Blocking Step: 10% Milk for 1 hour at room temperature.

See Abreview

Paraffin-embedded mouse backskin tissue ficed with Bouin's fixative, stained for ERp57 using ab13506 at 1/100 dilution in ICC/IF. Primary antibody was incubated for 1 hour at room temperature. Secondary antibody is a FITC-conjugated goat anti-mouse (green) incubated at 1/50 dilution for 1 hour at room temperature.

HaCaT (Human keratinocyte cell line) cells labeling ERp57 using ab13506 at 1/100 dilution in ICC.IF. Cells were fixed using 100% methanol for 10 minutes at -20°C and incubated with primary antibody for 1 hour at room temperature. Secondary antibody is a FITC-conjugated goat anti-mouse (green) incubated at 1/50 dilution for 1 hour at room temperature.

 

Overlay histogram showing HeLa cells stained with ab13506 (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. The cells were then incubated with the antibody (ab13506, 2µg/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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.