All lanes : Anti-160 kD Neurofilament Medium antibody [EPR23510-76] (ab254348) at 1/1000 dilution

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human nerve tissue lysate
Lane 3 : Human liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 102 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

This data was developed using ab254348, the same antibody clone in a different buffer formulation. 

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

The molecular weight is consistent to what has been described in the literature (PMID:19239416, PMID:27000625).

Negative control: liver (PMID:30541916).

Exposure time: 10 secs.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on human cerebellum. 

Secondary antibody only control: Secondary antibody is 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.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Negative control: No staining on mouse liver. 

Secondary antibody only control: Secondary antibody is 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.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of parental HEK-293T (EDWT04) and NEFM KO HEK-293T (ED040746) cells labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). 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 (Blue).

Confocal image showing cytoplasmic staining in parental HEK-293T cells and no staining in NEFM KO HEK-293T cells. 

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized wild-type HEK-293T (human embryonic kidney epithelial cell, Right)/ 160 kD Neurofilament Medium knockout HEK-293T cells (Left) labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 µg). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Positive staining on human wild-type HEK-293T cell line (ab255449), while no staining on human NEFM knockout HEK-293T cells (ab266741).

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

Positive staining on mouse cerebrum is observed. 

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488 )at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (blue).

Negative control: No staining on mouse liver (PMID: 30541916) is observed.

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

160 kD Neurofilament Medium was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 µg with ab254348 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10 µg.

Lane 2: ab254348 IP in mouse brain tissue lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254348 in mouse brain tissue lysate.

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

Exposure time: 3.25 secs.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

ELISA analysis using ab254348 at a range of 0-1000 ng/ml followed by a Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L）at 1/2500 dilution. Antigen concentration: 1000 ng/ml.

Antigens: Mouse 160 kD Neurofilament Medium.

All lanes : Anti-160 kD Neurofilament Medium antibody [EPR23510-76] (ab254348) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse cerebellum tissue lysate
Lane 3 : Mouse liver tissue lysate
Lane 4 : Rat brain tissue lysate
Lane 5 : Rat cerebellum tissue lysate
Lane 6 : Rat liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 102 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

This data was developed using ab254348, the same antibody clone in a different buffer formulation. 

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

The molecular weight is consistent to what has been described in the literature (PMID:19239416, PMID:27000625.

Negative control: liver (PMID:30541916).

Exposure times: Lane 1-3: 3.25 secs; Lane 4-6: 10 secs.

All lanes : Anti-160 kD Neurofilament Medium antibody [EPR23510-76] (ab254348) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : NEFM knockout HEK-293T whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 102 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

This data was developed using ab254348, the same antibody clone in a different buffer formulation. 

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

Lanes 1-3: Merged signal (red and green). Green - ab254348 observed at 160 kDa. Red - loading control ab8245 observed at 36 kDa.

ab254348 Anti-NEFM antibody [EPR23510-76] was shown to specifically react with NEFM in wild-type HEK-293T cells. Loss of signal was observed when the knockout cell line ab266741 (knockout cell lysate ab257103) was used. Wild-type and NEFM knockout samples were subjected to SDS-PAGE. ab254348 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at at 4°C overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on human cerebrum. 

Secondary antibody only control: Secondary antibody is 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.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on mouse cerebellum. 

Secondary antibody only control: Secondary antibody is 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.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 µg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on rat cerebellum. 

Secondary antibody only control: Secondary antibody is 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.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

Positive staining on rat cerebrum is observed. 

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488 )at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 µg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (blue).

Negative control: No staining on rat liver (PMID: 30541916) is observed.

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized mouse primary neuron cells labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 µg)/ Right compared with a Rabbit monoclonal IgG isotype control (ab172730) / Left.

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized rat primary neuron cells cells labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 µg)/ Right  compared with a Rabbit monoclonal IgG isotype control (ab172730) / Left.

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

This data was developed using ab254348, the same antibody clone in a different buffer formulation.

160 kD Neurofilament Medium was immunoprecipitated from 0.35 mg rat brain tissue lysate 10 µg with ab254348 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Rat brain tissue lysate 10 µg.

Lane 2: ab254348 IP in rat brain tissue lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254348 in rat brain tissue lysate.

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

Exposure time: 5.5 secs.