Recombinant Anti-CD44 antibody [EPR1013Y] (ab51037)

False colour image of Western blot: Anti-CD44 antibody [EPR1013Y] staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab51037 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-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lanes 1 - 4: Merged signal (red and green). Green - ab51037 observed at 80 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab51037 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 ab51037 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 5000 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.

Immunohistochemical analysis of paraffin-embedded human pancreatic cancer tissue labeling CD44 with ab51037 at 1/100 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). The sample was counter stained with hematoxylin.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Blocking and diluting buffer 5% NFDM/TBST

The expression of CD44 in MCF-7 is low (PMID: 25635866; PMID: 26005723). Jurkat does not express CD44 (PMID: 24127558).

ab51037 (1:100) showing positive staining in human Breast carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD44 with ab51037 at 1/100 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). The sample was counter stained with hematoxylin.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human skin tissue labeling CD44 with ab51037 at 1/100 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). The sample was counter stained with hematoxylin.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human cervical cancer tissue labeling CD44 with ab51037 at 1/100 dilution followed by goat anti-rabbit IgG H&L (HRP) (ab97051, 1/500). The sample was counter stained with hematoxylin.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab51037 (1:100) showing positive staining in human Glioma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab51037 (1:100) showing positive staining in human Thyroid gland carcinoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.