Immunohistochemical analysis of paraffin-embedded human testis tissue labeling Eg5 with 254298 at 1/1/4000 (0.17µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in spermatocytes of human testis is observed.
The section was incubated with ab254298 for 30 mins at room temperature.
The immunostaining 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labeling Eg5 with ab254298 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic and spindle (arrow) staining in NIH/3T3 cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

All lanes : Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

Lane 1 : Human testis tissue lysate
Lane 2 : Human tonsil tissue lysate
Lane 3 : Human lung tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/100000 dilution

Predicted band size: 119 kDa

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

The expression profile observed is consistent with what has been described in the literature (PMID:23857769; 27492783).

Negative control: Human lung (PMID: 27279560).

The minor bands beneath the target band may correspond to degradation.

Exposure time: 48 seconds.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labeling Eg5 with ab254298 at 1/600 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled 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.

Eg5 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug with ab254298 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254298 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 ug.

Lane 2: ab254298 IP in NIH/3T3 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254298 in NIH/3T3 whole cell lysate.

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

Exposure time: 3 minutes

Fresh lysates were used in this IP.

Eg5 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 ug with ab254298 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254298 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 ug.

Lane 2: ab254298 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254298 in HeLa whole cell lysate.

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

Exposure time: 3 minutes.

Fresh lysates were used in this IP.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Eg5 with ab254298 at 1/600 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled 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% Triton X-100 permeabilized HeLa cells labeling Eg5 with ab254298 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic and spindle (arrow) staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling Eg5 with ab254298 at 1/4000 (0.17µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Scattered cytoplasmic staining in human breast cancer cells is observed (PMID:29181100).
The section was incubated with ab254298 for 30 mins at room temperature.
The immunostaining 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Eg5 with 254298 at 1/4000 (0.17µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in human tonsil is observed (PMID:25277178).
The section was incubated with ab254298 for 30 mins at room temperature.
The immunostaining 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).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

All lanes : Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

Lane 1 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 2 : NCI-H1975 (human adenocarcinoma lung epithelial cell)
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell)
Lane 4 : 4T1 (mouse mammary gland carcinoma epithelial cell)

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 119 kDa

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

The expression profile observed is consistent with what has been described in the literature (PMID:23857769; 27492783).

Fresh lysates were tested in this WB.

The minor bands beneath the target band in lane 1 and lane 4 may correspond to degradation.

Exposure time: 15 seconds.

All lanes : Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

Lane 1 : Mouse testis tissue lysate
Lane 2 : Mouse brain tissue lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 119 kDa

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

Negative control: Mouse brain (PMID: 17974955).

The minor bands beneath the target band may correspond to degradation.

Exposure time: 48 seconds.