Recombinant Anti-ICAM1 antibody [EP1442Y] - Low endotoxin, Azide free (ab221777)

All lanes : Anti-ICAM1 antibody [EP1442Y] (ab53013) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ICAM1 CRISPR-Cas9 edited HeLa cell lysate
Lane 3 : A549 cell lysate
Lane 4 : Ramos cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

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 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 CRISPR-Cas9 edited lysate lane below 90 kDa (not observed by this antibody) 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 CRISPR-Cas9 edited 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.

This data was developed using ab53013, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling ICAM1 with purified ab53013 at 1/500 dilution (0.22 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This data was developed using ab53013, the same antibody clone in a different buffer formulation.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling ICAM1 with purified ab53013 at 1/500 dilution (0.22 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-ICAM1 antibody [EP1442Y] - BSA and Azide free (ab271852) at 1/1000 dilution

Lane 1 : Untreated HUVEC (Human umbilical vein endothelial cell) whole cell lysate
Lane 2 : HUVEC (Human umbilical vein endothelial cell) treated with 50ng/ml TNF-a for 24 hours whole cell lysate
Lane 3 : Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 58 kDa

The molecular mass observed is consistent with what has been described in the literatures (PMID: 29777158, 30082828, 31244919).

All lanes : Anti-ICAM1 antibody [EP1442Y] (ab53013) at 1/1000 dilution

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

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 58 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

This data was developed using ab53013, the same antibody clone in a different buffer formulation.

Lanes 1-4: Merged signal (red and green). Green - ab53013 observed at 90 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab53013 Anti-ICAM1 antibody [EP1442Y]  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°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.