Recombinant Anti-CD44 antibody [C44Mab-5] (ab264539)

All lanes : Anti-CD44 antibody [C44Mab-5] (ab264539)

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: 81 kDa
Observed band size: 75-80 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-CD44 antibody [C44Mab-5] staining at 1.226 µg/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab264539 was shown to bind specifically to CD44. A band was observed at 75-80 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD44 knockout cell line ab262515 (knockout cell lysate ab263938). To generate this image, wild-type and CD44 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes : Anti-CD44 antibody [C44Mab-5] (ab264539) at 1.226 µg/ml

Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 81 kDa
Observed band size: 82 kDa why is the actual band size different from the predicted?


Exposure time: 70 seconds

Blocking/diluting buffer and concentration: 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab264539).

Formalin-fixed, paraffin-embedded Human lung carcinoma tissue stained for CD44 using ab264539 at 0.253 µg/mL followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) in immunohistochemical analysis. Counterstained with Hematoxylin. Membranous staining on Human lung carcinoma. The section was incubated with ab264539 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Secondary antibody only control: Used PBS instead of the primary antibody, secondary antibody was a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Immunoprecipitation of CD44 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab264539 pre-coupled to prot.G-Sepharose beads. Samples were washed and processed for western blot with ab189524 at 1/2000. This data was kindly provided by the YCharOS Inc., an open science company with the mission of characterizing every commercially available antibody reagent. Abcam are working with YCharOS to support their mission of antibody characterisation using knock out cell lines.

Immunoprecipitation of CD44 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab264539 pre-coupled to prot.G-Sepharose beads. Samples were washed and processed for western blot with ab189524 at 1/2000. SM=10% starting material; UB=10% unbound fraction; IP=immunoprecipitate. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

Formalin-fixed, paraffin-embedded Human skin tissue stained for CD44 using ab264539 at 0.253 µg/mL followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) in immunohistochemical analysis. Counterstained with Hematoxylin. Membranous staining on human skin. The section was incubated with ab264539 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. 

Secondary antibody only control: Used PBS instead of the primary antibody, secondary antibody was a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Flow cytometric analysis of MDA-MB-231 (Human breast adenocarcinoma epithelial cell) cell line labeling CD44 (Red) using ab264539 at 1.055 µg/mL followed by Goat anti mouse IgG (Alexa Fluor^® 488, ab150113) at 1/2000 dilution. Mouse monoclonal IgG was used as the isotype control (Black). Cells without incubation with primary antibody and secondary antibody (Blue).

Gated on viable cells.