All lanes : Anti-NSUN2/SAKI antibody [EPR24140-93] (ab259941) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 4 : A431 (human epidermoid carcinoma 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 1/50000 dilution

Predicted band size: 86 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

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

The observed MW is consistent with what has described in the literature (PMID: 23604283, 25233213, 27447970, 31487418).

Exposure times: Lane 1: 15 seconds

Lane 2-4: 48 seconds.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling NSUN2/SAKI with ab259941 at 1/1000 dilution (0.503 μg/ml), followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and strong nuclear staining in HeLa cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling NSUN2/SAKI with ab259941 at 1/200 dilution (2.515 μg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Nuclear staining on human pancreas (PMID: 16713953). The section was incubated with ab259941 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 LeicaDS9800 (Bond^® Polymer Refine Detection).

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

NSUN2/SAKI was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab259941 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259941 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HEK-293 (human embryonic kidney epithelial cell) whole cell lysate 10 ug

Lane 2: ab259941 IP in HEK-293 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259941 in HEK-293 whole cell lysate

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

Exposure time: 15 seconds.

 

All lanes : Anti-NSUN2/SAKI antibody [EPR24140-93] (ab259941) at 1/1000 dilution

Lane 1 : ES-D3 (mouse embryonic mtipotent stem Cell) whole cell lysate
Lane 2 : Mouse spleen tissue lysate
Lane 3 : Rat cerebellum tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

Predicted band size: 86 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

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

The observed MW is consistent with what has described in the literature (PMID: 27306184).

Exposure times: Lane 1: 48 seconds

Lane 2, 3: 3 minutes.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labelling NSUN2/SAKI with ab259941 at 1/1000 (0.503 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) antibody at 1/1000 dilution (Green). Confocal image showing weak cytoplasmic and strong nuclear staining in NIH/3T3 cells.  ab195889 Anti-alpha Tubulin mouse monoclonal antibody - 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded mouse skin tissue labeling NSUN2/SAKI with ab259941 at 1/200 dilution (2.515 µg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Nuclear staining on mouse skin (PMID: 16713953). The section was incubated with ab259941 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 LeicaDS9800 (Bond^® Polymer Refine Detection).

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

NSUN2/SAKI was immunoprecipitated from 0.35 mg mouse spleen tissue lysate with ab259941 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259941 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse spleen tissue lysate 10 ug

Lane 2: ab259941 IP in Mouse spleen tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259941 in mouse spleen tissue lysate

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

Exposure time: 15 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling NSUN2/SAKI with ab259941 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) 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.

Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labeling NSUN2/SAKI with ab259941 at 1/200 dilution (2.515 µg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Nuclear staining on human colon carcinoma (PMID: 16713953). The section was incubated with ab259941 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 LeicaDS9800 (Bond^® Polymer Refine Detection).

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

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized ES-D3 (mouse embryonic multipotent stem cell) cells labelling NSUN2/SAKI with ab259941 at 1/50 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730)  isotype control (Black) 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.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling NSUN2/SAKI with ab259941 at 1/200 dilution (2.515 µg/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Nuclear staining on rat colon.The section was incubated with ab259941 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 LeicaDS9800 (Bond^® Polymer Refine Detection).

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