All lanes : Anti-Lamin A + Lamin C antibody [JOL2] (ab40567) at 1/200 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : LMNA knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 74 kDa
Observed band size: 74 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab40567 observed at 74 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab40567 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in LMNA knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and LMNA knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3pc Milk. Ab40567 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/200 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HeLa cells stained with ab40567 (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 (ab40567, 1/10 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1](ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue sections labelling Lamin A + C with ab40567.

Anti-Lamin A + Lamin C antibody [JOL2] (ab40567) at 1/200 dilution + HeLa whole cell lysate (10ug)

Secondary
HRP conjugated goat anti-mouse antibody

Developed using the ECL technique.

Performed under non-reducing conditions.

Predicted band size: 74 kDa
Observed band size: 64,73 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

See Abreview

Formalin-fixed, paraffin-embedded human skin tissue stained for Lamin A + C using ab40567 in immunohistochemical analysis. Slides were steamed in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C for 20 minutes and then let to stand at room temperature in a buffer for a further 20 minutes. They were then rinsed in 1X TBS with Tween (TBST) for 1 minute at room temperature.