Anti-GPNMB antibody [EPR18226-147]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GPNMB antibody [EPR18226-147] (AB188222)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized L-929 (mouse connective tissue fibroblast cell line) and EL4 (mouse lymphoma T lymphocyte cell line) cells labeling GPNMB with ab188222 at 1/1000 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 L-929 cells.

Negative control : EL4 (PMID : 19320736).

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)

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Flow Cytometry (Intracellular) - Anti-GPNMB antibody [EPR18226-147] (AB188222)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized EL4 (mouse lymphoma T lymphocyte cell line; left panel) and L-929 (mouse connective tissue fibroblast cell line; right panel) cell lines labeling GPNMB with ab188222 at 1/500 dilution (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 H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : EL4 (PMID : 19320736) (left panel).

• IP

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Immunoprecipitation - Anti-GPNMB antibody [EPR18226-147] (AB188222)

GPNMB was immunoprecipitated from 0.35 mg of L-929 (mouse connective tissue fibroblast cell line) whole cell lysate with ab188222 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab188222 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1 : L-929 whole cell lysate 10 μg (Input).

Lane 2 : ab188222 IP in L-929 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab188222 in L-929 whole cell lysate.

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

Exposure time : 1 minute.

All lanes:

Immunoprecipitation - Anti-GPNMB antibody [EPR18226-147] (ab188222)

Predicted band size: 64 kDa

Observed band size: 65 kDa,95 kDa

true

• WB

Supplier Data

Western blot - Anti-GPNMB antibody [EPR18226-147] (AB188222)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

Negative control : EL4 (PMID : 19320736).

All lanes:

Western blot - Anti-GPNMB antibody [EPR18226-147] (ab188222) at 1/5000 dilution

Lane 1:

L-929 (mouse connective tissue fibroblast cell line) whole cell lysate at 20 µg

Lane 2:

B16-F0 (mouse melanoma epithelial cell-like cell line) whole cell lysate at 20 µg

Lane 3:

EL4 (mouse lymphoma T lymphocyte cell line) whole cell lysate at 20 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,65 kDa,95 kDa

true

Exposure time: 15s