ab83504 staining ATF6 in HeLa cells. The cells were fixed with 4% PFA (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab83504 at 1/500 dilution and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). The secondary antibody (shown in red) was ab150084 Alexa Fluor® 594 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Lanes 1- 2: Merged signal (red and green). Green - ab83504 observed at 95 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab83504 was shown to react with ATF6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261800 (knockout cell lysate ab256841) was used. Wild-type HeLa and ATF6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab83504 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1 - 2: Merged signal (red and green). Green - ab83504 observed at 95 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab83504 was shown to recognize ATF6 in wild-type HAP1 cells along with additional cross reactive bands . No bands were observed when ATF6 knockout samples were used. Wild-type and ATF6 knockout samples were subjected to SDS-PAGE. Ab83504 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at a concentration of 1 ug/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

ATF6 has a predicted molecular weight of 75 kDa; however it has a number of potential glycosylation sites which may affect the migration of the protein (SwissProt data). We believe the bands observed at 75 and 100 kDa correspond to the full length nonglycosylated and glycosylated forms of ATF6.  Following Tunicamycin treatment to inhibit N-linked glycosylation we see the appearance of a partially deglycosylated form at 90 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab83504 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406