Human ICAM1 knockout HeLa cell lysate

• WB

Lab

Western blot - Human ICAM1 knockout HeLa cell lysate (AB256947)

Lane 1 : Wild-type HeLa cell lysate 20 μg
Lane 2 : ICAM1 knockout HeLa cell lysate 20 μg
Lane 3 : A549 cell lysate 20 μg
Lane 4 : Ramos cell lysate 20 μg
False colour image of Western blot : Anti-ICAM1 antibody [EPR4776] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109361 was shown to bind specifically to ICAM1. A band was observed at 90 kDa in wild-type HeLa cell lysates with no signal observed at this size in Icam1 knockout cell line ab261742 (knockout cell lysate ab256947). The band observed in the knockout lysate lane below 90 kDa is likely to represent a truncated form of ICAM1. 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 Icam1 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 °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-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-ICAM1 antibody [EPR4776] (<a href='/en-us/products/primary-antibodies/icam1-antibody-epr4776-ab109361'>ab109361</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ICAM1 knockout HeLa cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

Ramos cell lysate at 20 µg

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Human ICAM1 knockout HeLa cell lysate (AB256947)

Lane 1 : Wild-type HeLa cell lysate 20 μg
Lane 2 : ICAM1 knockout HeLa cell lysate 20 μg
Lane 3 : A549 cell lysate 20 μg
Lane 4 : Ramos cell lysate 20 μg
False colour image of Western blot : Anti-ICAM1 antibody [EP1442Y] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53013 was shown to bind specifically to ICAM1. A band was observed at 90 kDa (not observed by this antibody) in wild-type HeLa cell lysates with no signal observed at this size in Icam1 knockout cell line ab261742 (knockout cell lysate ab256947). The band observed in the knockout lysate lane below 90 kDa is likely to represent a truncated form of ICAM1. 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 Icam1 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 °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-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-ICAM1 antibody [EP1442Y] (<a href='/en-us/products/primary-antibodies/icam1-antibody-ep1442y-ab53013'>ab53013</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ICAM1 knockout HeLa cell lysate at 20 µg

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

Ramos cell lysate at 20 µg

Predicted band size: 57 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Human ICAM1 knockout HeLa cell lysate (AB256947)

Blocking and diluting buffer and concentration : Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ICAM1 knockout HeLa cell lysate
Lane 3 : Ramos cell lysate

Lanes 1-3 : Merged signal (red and green). Green - ab282575 observed at 90kDa. Red - loading control ab8245 observed at 36 kDa.

ab282575 Anti-ICAM1 antibody [EPR24639-3] was shown to react with ICAM1 in wild-type Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261742 (knockout cell lysate ab256947) was used. Wild-type and ICAM1 knockout samples were subjected to SDS-PAGE.

ab282575 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4^oC overnight 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 10000 dilution for 1 hour at room temperature before imaging.

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

ICAM1 knockout HeLa whole cell lysate

Lane 3:

Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

false

• WB

Unknown

Western blot - Human ICAM1 knockout HeLa cell lysate (AB256947)

Lane 1 : Wild-type HeLa cell lysate (20 ug)
Lane 2 : ICAM1 knockout HeLa cell lysate (20 ug)
Lane 3 : Raji cell lysate (20 ug)
Lane 4 : Ramos cell lysate (20 ug)

ab53013 was shown to specifically react with ICAM1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261742 (knockout cell lysate ab256947) was used. Wild-type and ICAM1 knockout samples were subjected to SDS-PAGE. ab53013 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4^oC 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.

false

• Sanger seq

Unknown

Sanger Sequencing - Human ICAM1 knockout HeLa cell lysate (AB256947)

Allele-2 : 1 bp insertion in exon 2

• Sanger seq

Unknown

Sanger Sequencing - Human ICAM1 knockout HeLa cell lysate (AB256947)

Allele-1 : Insertion of the selection cassette in exon 2