All lanes : Anti-GMEB1 antibody [EPR23056-62] (ab240646) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 3 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Lane 4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 63 kDa
Observed band size: 82 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 26 seconds; Lane 2: 125 seconds; Lane 3: 169 seconds; Lane 4: 3 minutes.

The weak lower bands below 75kDa could be degraded GMEB1 fragments (PMID:10386584).

All lanes : Anti-GMEB1 antibody [EPR23056-62] (ab240646) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with mouse GMEB1 (WT)(aa1-562) expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 63 kDa
Observed band size: 82 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 seconds.

The weak lower bands below 75kDa could be degraded GMEB1 fragments (PMID:10386584).

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling GMEB1 with ab240646 at 1/2000 dilution (0.288 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on rat spleen. The section was incubated with ab240646 for 10 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

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

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling GMEB1 with ab240646 at 1/1000 dilution (0.576 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on mouse spleen (PMID: 10692587) is observed. The section was incubated with ab240646 for 10 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

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

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling GMEB1 with ab240646 at 1/1000 dilution (0.576 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on human tonsil (PMID: 16497673) is observed. The section was incubated with ab240646 for 10 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

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

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling GMEB1 with ab240646 at 1/1000 dilution (0.576 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on human spleen (PMID: 16497673) is observed. The section was incubated with ab240646 for 10 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

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

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling GMEB1 with ab240646 at 1/1000 dilution (0.576 ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). Nuclear staining on human colon (PMID: 16497673) is observed. The section was incubated with ab240646 for 10 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer).

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

GMEB1 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab240646 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240646 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab240646 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab240646 in HeLa whole cell lysate

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

Exposure time: 3 min.

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293T (human embryonic kidney) transfected with mouse GMEB1 (WT)(aa1-562) expression vector containing a myc-His-tag® cells labelling GMEB1 with ab240646 at 1/500 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) (Left). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG (Left) or ab240646 (Right). Then stained with anti-myc tag conjugated to PE.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling GMEB1 with ab240646 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling GMEB1 with ab240646 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing mainly nuclear and weakly cytoplasmic staining in Neuro-2a cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is  ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling GMEB1 with ab240646 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000  dilution (Green). Confocal image showing mainly nuclear and weakly cytoplasmic staining in HeLa cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.