重组Anti-GPNMB抗体[EPR29063-10] (ab317325)

All lanes : Anti-GPNMB antibody [EPR29063-10] (ab317325) at 1/1000 dilution

Lane 1 : B16-F10 (Mouse skin melanoma) whole cell lysate
Lane 2 : B16-F0 (mouse melanoma epithelial cell-like) whole cell lysate
Lane 3 : EL4 (mouse lymphoma T lymphocyte) whole cell lysate
Lane 4 : 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 120,95 kDa why is the actual band size different from the predicted?


Exposure time: 37 seconds

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

Negative control: EL4(PMID: 19320736), 4T1(PMID: 20711474)

The identity of the lower MW band at approximately 15 kDa (in lane1 and 2) is unknown.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

All lanes : Anti-GPNMB antibody [EPR29063-10] (ab317325) at 1/1000 dilution

Lane 1 : Mouse testis tissue lysate
Lane 2 : Mouse spleen tissue lysate
Lane 3 : Rat testis tissue lysate
Lane 4 : Rat spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Observed band size: 120,65 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

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

Low expression: testis

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling GPNMB with ab317325 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spleen.
The section was incubated with ab317325 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse lung cancer tissue labeling GPNMB with ab317325 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse lung cancer (PMID: 29857071).
The section was incubated with ab317325 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling GPNMB with ab317325 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat spleen.
The section was incubated with ab317325 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling GPNMB with ab317325 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression: scattered staining on immune cells of mouse testis.
The section was incubated with ab317325 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling GPNMB with ab317325 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression: scattered staining on immune cells of rat testis.
The section was incubated with ab317325 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded A) B16-F0 (mouse melanoma epithelial-like cell) cell pellet; B) EL4 (mouse lymphoma T lymphocyte) cell pellet tissue labeling GPNMB with ab317325 at 1/5000 (0.104 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A) B16-F0 cell pellet; no staining on B) EL4 cell pellet.
The section was incubated with ab317325 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized B16-F0 (mouse melanoma epithelial-like cell) cells labelling GPNMB with ab317325 at 1/500 (1.036 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in B16-F0 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control: EL4 (PMID: 19320736).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.