Human CD58 knockout HeLa cell lysate

• WB

Lab

Western blot - Human CD58 knockout HeLa cell lysate (AB257880)

False colour image of Western blot : Anti-CD58 antibody [EPR24012-147] staining at 1/1000 dilution shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution shown in red. In Western blot ab275392 was shown to bind specifically to CD58. A band was observed at 55 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD58 knockout cell line ab265947 (knockout cell lysate ab257880). To generate this image wild-type and CD58 knockout HeLa 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 °. 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-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-CD58 antibody [EPR24012-147] (<a href='/en-us/products/primary-antibodies/cd58-antibody-epr24012-147-ab275392'>ab275392</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CD58 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CD58 knockout HeLa cell lysate (ab257880)

Lane 2:

Western blot - Human CD58 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cd58-knockout-hela-cell-line-ab265947'>ab265947</a>)

Lane 3:

Raji cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 28 kDa

Observed band size: 55 kDa

false

• WB

Lab

Western blot - Human CD58 knockout HeLa cell lysate (AB257880)

Lane 1 : Wild-type HeLa cell lysate (20 μg)

Lane 2 : CD58 knockout HeLa cell lysate (20 μg)

Lane 3 : HAP1 Wild-type cell lysate (20 μg)

Lane 4 : Daudi cell lysate (20 μg)

Lanes 1-4 : Merged signal (red and green). Green - ab196648 observed at 43 kDa. Red - loading control ab8245 observed at 37 kDa.

ab196648 Anti-CD58 antibody [EP15041] was shown to specifically react with CD58 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265947 (knockout cell lysate ab257880) was used. Wild-type and CD58 knockout samples were subjected to SDS-PAGE. ab196648 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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:

Western blot - Anti-CD58 antibody [EP15041] (<a href='/en-us/products/primary-antibodies/cd58-antibody-ep15041-ab196648'>ab196648</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CD58 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CD58 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cd58-knockout-hela-cell-line-ab265947'>ab265947</a>)

Lane 3:

HAP1 cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 28 kDa

Observed band size: 43 kDa

false

• Sanger seq

Unknown

Sanger Sequencing - Human CD58 knockout HeLa cell lysate (AB257880)

Allele-1 : 11 bp deletion in exon1

• Sanger seq

Unknown

Sanger Sequencing - Human CD58 knockout HeLa cell lysate (AB257880)

Allele-2 : 2 bp deletion in exon1