The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 75,90,100 kDa (predicted molecular weight: 75 kDa).
Transcription factor that acts during endoplasmic reticulum stress by activating unfolded protein response target genes. Binds DNA on the 5'-CCAC[GA]-3'half of the ER stress response element (ERSE) (5'-CCAAT-N(9)-CCAC[GA]-3') and of ERSE II (5'-ATTGG-N-CCACG-3'). Binding to ERSE requires binding of NF-Y to ERSE. Could also be involved in activation of transcription by the serum response factor.
Belongs to the bZIP family. ATF subfamily. Contains 1 bZIP domain.
The basic domain functions as a nuclear localization signal. The basic leucine-zipper domain is sufficient for association with the NF-Y trimer and binding to ERSE.
During unfolded protein response an approximative 50 kDa fragment containing the cytoplasmic transcription factor domain is released by proteolysis. The cleavage seems to be performed sequentially by site-1 and site-2 proteases. N-glycosylated. The glycosylation status may serve as a sensor for ER homeostasis, resulting in ATF6 activation to trigger the unfolded protein response (UPR). Phosphorylated in vitro by MAPK14/P38MAPK.
Endoplasmic reticulum membrane and Nucleus. Under ER stress the cleaved N-terminal cytoplasmic domain translocates into the nucleus.
Lane 1: Wild type HAP1 whole cell lysate (50 µg) Lane 2: ATF6 knockout HAP1 whole cell lysate (50 µg) 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.
ICC/IF image of ab83504 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 (ab83504, 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.
Western blot - Anti-ATF6 antibody (ab83504)
All lanes : Anti-ATF6 antibody (ab83504) at 1 µg/ml
Lane 1 : HeLa Whole Cell Lysate (untreated) Lane 2 : HeLa Whole Cell Lysate (treated with 2.37 uM Tunicamycin) Lane 3 : HeLa Whole Cell Lysate (treated with 23.7 uM Tunicamycin)
Lysates/proteins at 20 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution Developed using the ECL technique
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
Western blot - Anti-ATF6 antibody (ab83504)
Anti-ATF6 antibody (ab83504) at 1 µg/ml + MDA-MB-361 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique