All lanes : HRP Anti-CD44 antibody [EPR1013Y] (ab194989) at 1/2500 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 82 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab194989 observed at 80 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab194989 was shown to react with CD44 (HRP) 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 ab194989 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 2500 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.

HRP Anti-CD44 antibody [EPR1013Y] (ab194989) at 1/2500 dilution + TF-1 Whole Cell Lysate at 10 µg

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 82 kDa
Observed band size: 82 kDa


Exposure time: 20 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab194989 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.