All lanes : Anti-RALA antibody [EPR6468] (ab126627) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RALA knockout HeLa cell lysate
Lane 3 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 24 kDa

Lanes 1-3: Merged signal (red and green). Green - ab126627 observed at 25 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab126627 Anti-RALA antibody [EPR6468] was shown to specifically react with RALA in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265092 (knockout cell lysate ab258165) was used. Wild-type and RALA knockout samples were subjected to SDS-PAGE. ab126627 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 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-RALA antibody [EPR6468] (ab126627) at 1/1000 dilution

Lane 1 : MCF7 cell lysate
Lane 2 : MOLT4 cell lysate
Lane 3 : BxPC3 cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 24 kDa

Overlay histogram showing MCF7 cells stained with ab126627 (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 (ab126627, 1/10000 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 MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab126627, at 1/100, staining RALA in formalin fixed, paraffin embedded Human breast carcinoma tissue by Immunohistochemistry.

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