Anti-NP-I antibody [EPR25683-64] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling NP-I with ab289966 at 1/2000 followed by LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on human cerebrum. The section was incubated with ab289966 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS instead of primary antibody, followed by LeicaDS9800 (Bond™, Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labelling NP-I with ab289966 at 1/2000 followed by LeicaDS9800 (Bond™, Polymer Refine Detection). Negative control : no staining on human heart. The section was incubated with ab289966 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling NP-I with ab289966 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

Lab

Immunoprecipitation - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289990, the same antibody clone in a different buffer formulation.

NP-I was immunoprecipitated from HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab2568364 at 1/30 dilution (2ug in 0.07mg lysates). Western blot was performed on the immunoprecipitate using anti npi antibody epr2568364 immunoprecipitation hela.jpg at 1/5000 dilution. Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, light chain specific was used at 1/100000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 2ug

Lane 2 : ab2568364 IP in HeLa whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab289966 in HeLa whole cell lysate

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

Exposure time : 3.25 seconds

The 38KDa band might be NPTX1 fragmemts (PMID : 29501530).

All lanes:

Immunoprecipitation - Anti-NP-I antibody [EPR25683-64] (<a href='/en-us/products/primary-antibodies/np-i-antibody-epr25683-64-ab289966'>ab289966</a>)

Predicted band size: 47 kDa

Observed band size: 38 kDa,50 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling NP-I with ab289966 at 1/2000 followed by LeicaDS9800 (Bond™, Polymer Refine Detection). Positive staining on rat cerebrum. The section was incubated with ab289966 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS instead of primary antibody, followed by LeicaDS9800 (Bond™, Polymer Refine Detection).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling NP-I with ab289966 at 1/2000 followed by LeicaDS9800 (Bond™, Polymer Refine Detection) was used. Positive staining on mouse cerebrum. The section was incubated with ab289966 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS instead of primary antibody, followed by LeicaDS9800 (Bond™, Polymer Refine Detection).

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

• IP

Lab

Immunoprecipitation - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

NP-I was immunoprecipitated from Mouse hypothalamus tissue lysate with ab289966 at 1/30 dilution (2ug in 0.175mg lysates). Western blot was performed on the immunoprecipitate using ab289966 at 1/5000 dilution. Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, light chain specific was used at 1/100000 dilution.

Lane 1 : Mouse hypothalamus tissue lysate 5 ug

Lane 2 : ab289966 IP in Mouse hypothalamus tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab289966 in Mouse hypothalamus tissue lysate

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

Exposure time : 3.25 seconds

The 38KDa band might be NPTX1 fragmemts (PMID : 29501530).

All lanes:

Immunoprecipitation - Anti-NP-I antibody [EPR25683-64] (<a href='/en-us/products/primary-antibodies/np-i-antibody-epr25683-64-ab289966'>ab289966</a>)

Predicted band size: 47 kDa

Observed band size: 38 kDa,50 kDa

false

• WB

Lab

Western blot - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

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

Exposure time : 10 seconds

All lanes:

Western blot - Anti-NP-I antibody [EPR25683-64] (<a href='/en-us/products/primary-antibodies/np-i-antibody-epr25683-64-ab289966'>ab289966</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 16 µg

Lane 2:

HeLa transfected with siRNA specifically targeti NPTX1 whole cell lysate at 16 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/100000 dilution

Lanes 1 - 2:

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: 47 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

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

NPTX1 is a glycosylated protein and can be deglycosylated by Protein Deglycosylation MIX II.

Negative control : human heart (PMID : 7695898)

Exposure time : 7.75 seconds

All lanes:

Western blot - Anti-NP-I antibody [EPR25683-64] (<a href='/en-us/products/primary-antibodies/np-i-antibody-epr25683-64-ab289966'>ab289966</a>) at 1/1000 dilution

Lane 1:

Human hypothalamus tissue lysate at 20 µg

Lane 2:

Human heart tissue lysate at 20 µg

Lane 3:

Untreated Human hypothalamus tissue lysate at 20 µg

Lane 4:

Human hypothalamus tissue lysate treated with Protein Deglycosylation MIX II at 20 µg

Secondary

Lanes 1 - 2:

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

Lanes 3 - 4:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 47 kDa,50 kDa

false

• WB

Lab

Western blot - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 29501530)

Exposure time : 15 seconds.

All lanes:

Western blot - Anti-NP-I antibody [EPR25683-64] (<a href='/en-us/products/primary-antibodies/np-i-antibody-epr25683-64-ab289966'>ab289966</a>) at 1/1000 dilution

Lane 1:

Mouse hypothalamus tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

Rat brain tissue lysate at 20 µg

Lane 4:

Rat striatum 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: 47 kDa

Observed band size: 38 kDa,50 kDa

false

• WB

Lab

Western blot - Anti-NP-I antibody [EPR25683-64] - BSA and Azide free (AB289990)

This data was developed using ab289966, the same antibody clone in a different buffer formulation.

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

Exposure times : Lane 1 : 1 seconds; Lane 2 : 180 seconds

All lanes:

Western blot - Anti-NP-I antibody [EPR25683-64] (<a href='/en-us/products/primary-antibodies/np-i-antibody-epr25683-64-ab289966'>ab289966</a>) at 1/1000 dilution

Lane 1:

His-tagged human NPTX1 recombinant protein, 10 ng

Lane 2:

His-tagged human NPTX2 recombinant protein, 10 ng

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 47 kDa

Observed band size: 50 kDa

false