All lanes : Anti-Rel B antibody [EP614Y] (ab33907) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RELB knockout HeLa cell lysate
Lane 3 : Raji cell lysate
Lane 4 : LnCap cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 62 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab33907 observed at 70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab33907 was shown to react with Rel B in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265948 (knockout cell lysate ab257635) was used. Wild-type HeLa and RELB knockout HeLa cell lysates were subjected to SDS-PAGE. ab33907 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 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.

All lanes : Anti-Rel B antibody [EP614Y] (ab33907) at 1/2000 dilution (purified)

Lane 1 : Raji cell lysate
Lane 2 : Daudi 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: 62 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunofluorescence staining of Raji cells with purified ab33907 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4 % PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab33907 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

ab33907 (purified) at 1/20 immunoprecipitating Rel B in 10 μg Daudi cell lysate (Lanes 1 and 2, observed at 70 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730). For western blotting, HRP Veriblot for IP (ab131366) was used for detection (1/1000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

Overlay histogram showing Raji cells fixed in 80% methanol and stained with purified ab33907 at a dilution of 1/70 (red line). The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Anti-Rel B antibody [EP614Y] (ab33907) at 1/20000 dilution (unpurified) + Raji Cell Lysate

Predicted band size: 62 kDa
Observed band size: 62 kDa

Overlay histogram showing Raji cells stained with unpurified ab33907 (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 (ab33907, 1/1000 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) (0.1μg/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. This antibody gave a positive signal in Raji cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.