Anti-CD44 antibody [EPR18668]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human kidney.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labeling CD44 with ab189524 at 1/8000 dilution (0.099 μg/ml), followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining on human endometrial carcinoma. The section was incubated with ab189524 at 4°C overnight. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer (ab93684) pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human breast cancer [PMID : 15867228].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human breast [PMID : 20103682].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on human tonsil.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-CD44 antibody [EPR18668] (AB189524)

CD44v6 was immunoprecipitated from 1 mg of A549 (Human lung carcinoma cell line) whole cell lysate with ab189524 at 1/25 dilution. Western blot was performed from the immunoprecipitate using ab189524 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : A549 whole cell lysate 10 μg (Input).

Lane 2 : ab189524 IP in A549 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab189524 in A549 whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-CD44 antibody [EPR18668] (ab189524)

Predicted band size: 81 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on mouse spleen.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on mouse colon.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on rat spleen.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded mouse stomach tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on mouse stomach.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [EPR18668] (AB189524)

Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling CD44 with ab189524 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on rat stomach.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-CD44 antibody [EPR18668] (AB189524)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD44 antibody [EPR18668] (ab189524) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/10000 dilution

Predicted band size: 81 kDa

Observed band size: 82 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-CD44 antibody [EPR18668] (AB189524)

Blocking/Dilution buffer : 5% NFDM/TBST.

Due to high sequence homology between CD44 isoforms, within the immunogen region, this antibody cross reacts with most isoforms of CD44. The staining pattern is consistent with reference (PMID 19507218).

All lanes:

Western blot - Anti-CD44 antibody [EPR18668] (ab189524) at 1/2000 dilution

Lane 1:

Human thymus lysate at 10 µg

Lane 2:

Human skin lysate at 10 µg

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 10 µg

Lane 4:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 10 µg

Secondary

Lanes 1 - 2:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/10000 dilution

Lanes 3 - 4:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 81 kDa

Observed band size: 82 kDa

false

Exposure time: 1s

• WB

Collaborator

Western blot - Anti-CD44 antibody [EPR18668] (AB189524)

ab189524 was shown to react with CD44 in wild-type HAP1 cells in Western blot with loss of signal observed in a CD44 knockout cell line. Wild-type HAP1 and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab189524 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2ug/mL before imaging. 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.

All lanes:

Western blot - Anti-CD44 antibody [EPR18668] (ab189524) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

CD44 knockout HAP1 cell lysate at 20 µg

Predicted band size: 81 kDa

false

• WB

Lab

Western blot - Anti-CD44 antibody [EPR18668] (AB189524)

False colour image of Western blot : Anti-CD44 antibody [EPR18668] 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, ab189524 was shown to bind specifically to CD44. A band was observed at 70-85 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 [EPR18668] (ab189524) 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: 70-85 kDa

false

• WB

Lab

Western blot - Anti-CD44 antibody [EPR18668] (AB189524)

Lanes 1 - 4 : Merged signal (red and green). Green - ab189524 observed at 80 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab189524 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 ab189524 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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 [EPR18668] (ab189524) 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

Supplier Data

Western blot - Anti-CD44 antibody [EPR18668] (AB189524)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1, 2, 3, 4, 5 and 6 : 5 seconds; Lane 7, 8, 9 and 10 : 1 second.

Lanes 1 - 6:

Western blot - Anti-CD44 antibody [EPR18668] (ab189524) at 1/1000 dilution

Lanes 7 - 10:

Western blot - Anti-CD44 antibody [EPR18668] (ab189524) at 1/20000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse spleen lysate at 10 µg

Lane 3:

Rat brain lysate at 10 µg

Lane 4:

Rat heart lysate at 10 µg

Lane 5:

Rat kidney lysate at 10 µg

Lane 6:

Rat spleen lysate at 10 µg

Lane 7:

C6 (Rat glial tumor cell line) whole cell lysate

Lane 8:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate

Lane 9:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate

Lane 10:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 81 kDa

Observed band size: 82 kDa

false