Anti-CD44 antibody [SP37]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [SP37] (AB101531)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human esophageal carcinoma tissue sections labeling CD44 with Purified ab101531 at 1/100 dilution (1.03 µg/ml). Heat mediated antigen retrieval was performed Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 20mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [SP37] (AB101531)

Formalin-fixed, paraffin-embedded human esophageal carcinoma tissue stained for CD44 with ab101531 at a 1/100 dilution in immunohistochemical analysis.

• Flow Cyt

Lab

Flow Cytometry - Anti-CD44 antibody [SP37] (AB101531)

Flow cytometric analysis of Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Left) / A549 (human lung carcinoma epithelial cell, Right) cells labelling CD44 with ab101531 at 1/50 dilution (1µg) (Red) followed by a Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) secondary antibody used at a 1/5000 dilution compared with a Rabbit monoclonal IgG (ab172730) ( Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Gated on viable cells.
Negative control : Jurkat.

• WB

Lab

Western blot - Anti-CD44 antibody [SP37] (AB101531)

False colour image of Western blot : Anti-CD44 antibody [SP37] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab101531 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.

All lanes:

Western blot - Anti-CD44 antibody [SP37] (ab101531) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CD44 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CD44 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cd44-knockout-hela-cell-line-ab262515'>ab262515</a>)

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

LNCaP cell lysate at 20 µg

Predicted band size: 81 kDa

Observed band size: 75-80 kDa

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• WB

Lab

Western blot - Anti-CD44 antibody [SP37] (AB101531)

Lanes 1 - 4 : Merged signal (red and green). Green - ab101531 observed at 80 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab101531 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 ab101531 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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.

All lanes:

Western blot - Anti-CD44 antibody [SP37] (ab101531) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD44 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cd44-knockout-hela-cell-line-ab262515'>ab262515</a>)

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg

Predicted band size: 81 kDa

Observed band size: 80 kDa

false

• WB

Unknown

Western blot - Anti-CD44 antibody [SP37] (AB101531)

All lanes:

Western blot - Anti-CD44 antibody [SP37] (ab101531) at 1/25 dilution

All lanes:

HeLa cell lysate

Predicted band size: 41 kDa,81 kDa

Observed band size: 80 kDa

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