Anti-Eg5 antibody [EPR23276-52]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Eg5 antibody [EPR23276-52] (AB254298)

Intracellular 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

• IP

Unknown

Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

All lanes:

Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (ab254298)

Predicted band size: 119 kDa

false

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Eg5 antibody [EPR23276-52] (AB254298)

Intracellular 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

• IP

Unknown

Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

All lanes:

Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (ab254298)

Predicted band size: 119 kDa

false

• WB

Lab

Western blot - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

All lanes:

Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

Human lung tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/100000 dilution

Predicted band size: 119 kDa

false

• WB

Lab

Western blot - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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:

Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 20 µg

Lane 2:

NCI-H1975 (human adenocarcinoma lung epithelial cell) at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) at 20 µg

Lane 4:

4T1 (mouse mammary gland carcinoma epithelial cell) 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: 119 kDa

false

• WB

Lab

Western blot - Anti-Eg5 antibody [EPR23276-52] (AB254298)

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.

All lanes:

Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse brain tissue 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: 119 kDa

false