Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling NPTX2 with ab277523 at 1/2000 (0.229 ug/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Positive staining on mouse cerebrum (PMID:31772567). The section was incubated with ab277523 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) was used.

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

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling NPTX2 with ab277523 at 1/2000 (0.229 ug/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection) was used. Positive staining on human cerebrum. The section was incubated with ab277523 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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 100% Methanol permeabilized Beta-TC-6 cells labelling NPTX2 with ab277523 at 1/50 (9.16 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in Beta-TC-6 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.

All lanes : Anti-NPTX2 antibody [EPR24020-38] (ab277523) at 1/1000 dilution

Lane 1 : Beta-TC-6 (mouse pancreas insulinoma beta cell) whole cell lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Mouse testis tissue lysate
Lane 4 : Mouse kidney tissue lysate
Lane 5 : Mouse lung tissue lysate
Lane 6 : Rat brain tissue lysate
Lane 7 : Rat testis 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: 47 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

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

NPTX2 is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID: 8786423, 28440221).

Negative: mouse kidney, mouse lung (PMID: 8530029, 8786423).

Exposure time: 15 seconds.

 

NPTX2 was immunoprecipitated from 0.35 mg Mouse brain tissue lysate  with ab277523 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab277523 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10 ug

Lane 2: ab277523 IP in Mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab277523 in mouse brain tissue lysate

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

Exposure time: 10 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Beta-TC-6 (Mouse pancreas insulinoma beta cell) cells labelling NPTX2 with ab277523 at 1/50 dilution (1ug)(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 pancreas tissue labeling NPTX2 with ab277523 at 1/2000 (0.229 ug/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Positive staining on islet of human pancreas. The section was incubated with ab277523 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.

All lanes : Anti-NPTX2 antibody [EPR24020-38] (ab277523) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Human pancreas tissue lysate
Lane 3 : Human testis tissue lysate
Lane 4 : Human kidney tissue lysate
Lane 5 : 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: 47 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

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

NPTX2 is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID: 8786423, 28440221).

Negative: human kidney, human lung (PMID: 8530029, 8786423).

Exposure times: Lane 1: 15 secondsLane 2-5: 3 minutes.

 

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling NPTX2 with ab277523 at 1/2000 (0.229 ug/ml) followed by a ready to use LeicaDS9800 (Bond^® Polymer Refine Detection). Positive staining on rat cerebrum. The section was incubated with ab277523 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.

NPTX2 was immunoprecipitated from 0.35 mg Human brain tissue lysate  with ab277523 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab277523 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Human brain tissue lysate 10 ug

Lane 2: ab277523 IP in Human brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab277523 in human brain tissue lysate

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

Exposure time: 10 seconds.

 

Anti-NPTX2 antibody [EPR24020-38] (ab277523) at 1/1000 dilution + SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

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

Predicted band size: 47 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

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

NPTX2 is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID: 8786423, 28440221).

Exposure time: 15 seconds.