All lanes : Anti-CD44 antibody [MEM-263] (ab9524) at 2 µg/ml

Lane 1 : MOLT-4 cells (human T lymphoblast; acute lymphoblastic leukemia)
Lane 2 : HeLa cells (human epithelial cell line from cervix adenocarcinoma)

Performed under non-reducing conditions.

Human peripheral blood lymphocytes stained with ab9524 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab9524, 0.1μg/1x10⁶ cells) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.

All lanes : Anti-CD44 antibody [MEM-263] (ab9524) at 2 µg/ml

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.

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

Lanes 1 - 4: Merged signal (red and green). Green - ab9524 observed at 80 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab9524 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 and CD44 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab9524 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 2 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

IHC image of CD44 staining in Human Skin Melanoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab9524, 0.5 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

 

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Western blotting of HPB-ALL cell lysate (non-reduced sample) stained by ab9524.

Western blotting of HPB-ALL cell lysate (non-reduced sample) stained by ab9524.