Anti-Beclin 1抗体[OTI4A10] (ab118148)

All lanes : Anti-Beclin 1 antibody [OTI4A10] (ab118148) at 1/2000 dilution

Lane 1 : Wild-type Jurkat cell lysate
Lane 2 : BECN1 CRISPR-Cas9 edited Jurkat cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 52 kDa

False colour image of Western blot: Anti-Beclin 1 antibody [OTI4A10] staining at 1/2000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab118148 was shown to bind specifically to Beclin 1. A band was observed at 52 kDa in wild-type Jurkat cell lysates with no signal observed at this size in BECN1 CRISPR-Cas9 edited cell line ab283778 (BECN1 CRISPR-Cas9 edited cell lysate ab283792). The band observed in the CRISPR-Cas9 edited lysate lane above 52 kDa is likely to represent Beclin 1 with an insertion. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and BECN1 CRISPR-Cas9 edited Jurkat cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes : Anti-Beclin 1 antibody [OTI4A10] (ab118148) at 1/1000 dilution

Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : BECN1 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab118148 observed at 60 kDa. Red - loading control, ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab118148 was shown to react with Beclin 1 in wild-type HeLa cells in western blot. Loss of signal was observed when BECN1 knockout sample was used. Wild-type HeLa and BECN1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in non-mammalian (TBS-based) blocking solution before incubation with ab118148 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Beclin 1 antibody [OTI4A10] (ab118148) at 1/2000 dilution

Lane 1 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with pCMV6-ENTRY control
Lane 2 : HEK-293T cells transfected with pCMV6-ENTRY Beclin 1

Lysates/proteins at 5 µg per lane.

Predicted band size: 52 kDa