All lanes : Anti-Eg5 antibody [EPR23277-7] (ab272220) 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 secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 119 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

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: 70 seconds.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Eg5 with ab272220 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in germinal center of human tonsil is observed (PMID:25277178). The section was incubated with ab272220 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 HeLa cells labeling Eg5 with ab272220 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing spindle fibre and cytoplasmic staining in HeLa cell line. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/1000 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 dilution.

Flow cytometric analysis of 80% methanol fixed 0.1% Tween-20 permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Eg5 with ab272220 at 1/50 dilution (0.1µg)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were pre-treated with 20µg/ml RNaseA for 30min to minimize the binding between Propidium Iodide (PI) and RNA. Then intracellularly stained with ab272220 and PI. Propidium Iodide Flow Cytometry Kit (ab139418) was used for RNaseA treatment and PI staining.

All lanes : Anti-Eg5 antibody [EPR23277-7] (ab272220) at 1/1000 dilution

Lane 1 : Mouse testis tissue lysate
Lane 2 : Rat testis tissue lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 119 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

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

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

Exposure time: Lanes 1-2: 10 seconds; Lane 3: 3 seconds;Lane 4: 26 seconds.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Eg5 with ab272220 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in spermatocytes and spermatogonnia of mouse testis is observed. The section was incubated with ab272220 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 ab272220 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing spindle fibre and cytoplasmic staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/1000 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 dilution.

All lanes : Anti-Eg5 antibody [EPR23277-7] (ab272220) 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), whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : 4T1 (mouse mammary gland carcinoma epithelial cell), whole cell lysate
Lane 5 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 7 : AR42J (rat pancreatic tumor epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 119 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

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

Fresh lysates were used in this WB.

Exposure times: Lane 1: 8 seconds; Lanes 2-7: 48 seconds.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling Eg5 with ab272220 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in spermatocytes and spermatogonnia of rat testis is observed. The section was incubated with ab272220 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 breast cancer tissue labeling Eg5 with ab272220 at 1/500 dilution 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 ab272220 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.