All lanes : Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : DAXX knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab32140 was shown to react with Daxx in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265233 (knockout cell lysate ab257408) was used. Wild-type HeLa and DAXX 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. ab32140 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 dilution 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.

ab32140 stained HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32140 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

All lanes : Anti-Daxx antibody [E94] (ab32140)

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : DAXX knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 81 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab32140 observed at 100 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32140 was shown to specifically react with Daxx in wild-type HAP1 cells as signal was lost in DAXX knockout cells. Wild-type and DAXX knockout samples were subjected to SDS-PAGE. ab32140 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Daxx antibody [E94] (ab32140) at 0.271 µg/ml

Lane 1 : PC-12(Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 2 : NIH/3T3(Mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 81 kDa


Exposure time: 29 seconds

Blocking and diluting buffer: 5% NFDM/TBST.

Overlay histogram showing HeLa cells stained with ab32140 (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 followed by the antibody (ab32140, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Anti-Daxx antibody [E94] (ab32140) at 1/5000 dilution + Hela cell lysate

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?
Additional bands at: 48 kDa. We are unsure as to the identity of these extra bands.

Ab32140, at a dilution of 1/50, staining Daxx in paraffin embedded human stomach adenocarcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.