Anti-GPNMB antibody [EPR22011-47]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-GPNMB antibody [EPR22011-47] (AB235873)

ab235873 was shown to react with GPNMB in wild-type U-87 MG cells in immunocytochemistry with loss of signal observed in a GPNMB siRNA knockdown cell line. Wild-type and siRNA knockdown 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 with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab235873 at 1/250 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (siRNA knockdown) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and siRNA knockdown 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).

This data was 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

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GPNMB antibody [EPR22011-47] (AB235873)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SK-MEL-28 (human malignant melanoma) and HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GPNMB with ab235873 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining in SK-MEL-28 cell line.

Negative control : HeLa （PMID : 20056711.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-GPNMB antibody [EPR22011-47] (AB235873)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell, Left panel) andSK-MEL-28 (human malignant melanoma, Right panel) labeling GPNMB with ab235873 at 1/500 (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : HeLa (PMID : 20056711).

• WB

Supplier Data

Western blot - Anti-GPNMB antibody [EPR22011-47] (AB235873)

Blocking/Dilution buffer : 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literature (PMID : 19320736).

Negative control : HeLa (PMID : 20056711).

All lanes:

Western blot - Anti-GPNMB antibody [EPR22011-47] (ab235873) at 1/1000 dilution

Lanes 1 - 2:

SK-MEL-28 (human malignant melanoma) whole cell lysate at 10 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 10 µg

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: 64 kDa

Observed band size: 120 kDa,95 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-GPNMB antibody [EPR22011-47] (AB235873)

ab235873 was shown to react with GPNMB in wild-type U-87 MG cells in Western blot with loss of signal observed in a GPNMB siRNA knockdown cell line. Cell lysates from wild-type U-87 MG transfected with either scrambled siRNA or GPNMB siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab235873 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was 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-GPNMB antibody [EPR22011-47] (ab235873) at 1/500 dilution

Lane 1:

Wild-type U-87 MG transfected with scrambled siRNA control lysate at 60 µg

Lane 2:

U-87 MG transfected with siRNA specifically targeting GPNMB cell lysate at 60 µg

false