All lanes : Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023) at 1/1000 dilution

Lane 1 : A431 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : KRT8 knockout HeLa

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution

Predicted band size: 53 kDa
Additional bands at: 37 kDa (possible Loading Control)

Lanes 1 - 4: Merged signal (red and green). Green - ab9023 observed at 55 kDa. Red - loading control, ab181602 observed at 37 kDa.  

 ab9023 was shown to react with Cytokeratin 8 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255400 (knockout cell lysate ab263785) was used. Wild-type and Cytokeratin 8 knockout samples were subjected to SDS-PAGE. ab9023 and Anti-GAPDH antibody EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-Cytokeratin 8 antibody [EP1628Y] - Cytoskeleton Marker (ab53280) at 1/10000 dilution

Lane 1 : A431 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : KRT8 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

Predicted band size: 53 kDa
Additional bands at: 37 kDa (possible Loading Control)

Lanes 1 - 4: Merged signal (red and green). Green - ab53280 observed at 55 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab53280 was shown to react with Cytokeratin 8 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255400 (knockout cell lysate ab263785) was used. Wild-type and Cytokeratin 8 knockout samples were subjected to SDS-PAGE. ab53280 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 (For unpurified use at 1/25,000 - 1/50,000) 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.

 

 

Allele-1: 4 bp deletion in exon 2.

 

Allele-2: 2 bp deletion in exon 2.

 

Allele-3: 1 bp insertion in exon 2.