Formalin-fixed, paraffin-embedded human head and neck squamous cell carcinoma tissue stained for CD44 using ab243894 at 1/500 dilution in immunohistochemical analysis. A HRP-conjugated goat anti-rabbit antibody was used as the secondary. DAB staining.

All lanes : Anti-CD44 antibody [BLR038F] (ab243894) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : Knockout CD44 HeLa cell lysate
Lane 3 : A549 cell lysate
Lane 4 : LnCAP cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab243894 observed at 80 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab243894 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab243894 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

CD44 was immunoprecipitated from 1.0 mg HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab243894 at 20 µl per reaction. Western blot was performed on the immunoprecipitate using ab243894 at 1/1000 dilution.

Lane 1: ab243894 IP in HeLa whole cell lysate.

Lane 2: Control IgG in HeLa whole cell lysate.

Detection: Chemiluminescence with an exposure time of 3 seconds.

Western blot analysis using ab243894 at 1/1000 dilution.

Lane 1: HeLa whole cell lysate (50 µg).

Lane 2: 786-O whole cell lysate (50 µg).

Lane 3: HEK-293T whole cell lysate (50 µg).

Lane 4: Jurkat-3 whole cell lysate (50 µg).

Lane 5: A549 whole cell lysate (50 µg).

Lane 6: MCF7 whole cell lysate (50 µg).

Lane 7: HepG2 whole cell lysate (50 µg).

Lane 8: KG-1 whole cell lysate (50 µg).

Lane 9: RPMI 8226 whole cell lysate (50 µg).

Lane 10: MOLT-4 whole cell lysate (50 µg).

A HRP-conjugated goat anti-rabbit IgG antibody was used as the secondary. Detection: chemiluminescence with an exposure time of 3 seconds.

Formalin-fixed, paraffin-embedded HCT 116 (human colorectal carcinoma cell line) cells labeling CD44 using ab243894 at 1/500 dilution in ICC analysis. A HRP-conjugated goat-anti rabbit IgG was used as the secondary. DAB staining.