Anti-CD44 antibody [Hermes-3]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [Hermes-3] (AB254530)

Immunohistochemical analysis of paraffin-embedded human skin tissue labeling CD44 with ab254530 at 0.107μg/ml, followed by ready to use secondary. Membranous staining on human skin is observed. The section was incubated with ab254530 for 5 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, followed by ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-CD44 antibody [Hermes-3] (AB254530)

ab254530 was shown to react with CD44 in wild-type HAP1 cells in Immunocytochemistry with loss of signal observed in a CD44 knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked. The cells were then incubated with ab254530 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a secondary antibody to (Alexa Fluor^® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880). 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD44 antibody [Hermes-3] (AB254530)

ab254530 staining CD44 in wild-type HeLa cells (top panel) and CD44 knockout HeLa cells (ab262515)(bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab254530 at 0.4μg/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor^® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD44 antibody [Hermes-3] (AB254530)

Immunohistochemical analysis of paraffin-embedded human bladder carcinoma tissue labeling CD44 with ab254530 at 0.107μg/ml, followed by ready to use secondary. Membranous staining on human bladder carcinoma is observed. The section was incubated with ab254530 for 5 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, followed by ready to use secondary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• IP

Collaborator

Immunoprecipitation - Anti-CD44 antibody [Hermes-3] (AB254530)

Immunoprecipitation of CD44 in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 1.0 μg of ab254530 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.

All lanes:

Immunoprecipitation - Anti-CD44 antibody [Hermes-3] (ab254530)

Predicted band size: 81 kDa

false

• ELISA

Supplier Data

ELISA - Anti-CD44 antibody [Hermes-3] (AB254530)

ELISA - Anti-CD44 antibody [Hermes-3] (ab254530).

Antigen : Human CD44.

ab254530 used at 100-1000μg/ml.

Secondary is an Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) used at a 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-CD44 antibody [Hermes-3] (AB254530)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD44 antibody [Hermes-3] (ab254530) at 0.536 µg/mL

Lane 1:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 81 kDa

false

Exposure time: 1s

• WB

Collaborator

Western blot - Anti-CD44 antibody [Hermes-3] (AB254530)

ab254530 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 ab254530 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with goat anti-mouse HRP secondary antibodies at 0.3ug/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 [Hermes-3] (ab254530) 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