Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labelling WFS1 with ab259362 at 1/5000 dilution (0.093 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on mouse pancreatic islet (PMID: 15994758). The section was incubated with ab259362 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 cells labelling WFS1 with ab259362 at 1/50 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 HepG2 cells is observed. 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 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse pancreas tissue labeling WFS1 with ab259362 at 1/50 (9.28 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on mouse pancreatic islets is observed. The nuclear counterstain was DAPI (Blue).

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).

All lanes : Anti-WFS1 antibody [EPR23801-91] (ab259362) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 100 kDa
Observed band size: 300, 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 been described in the literature (PMID: 12913071). 

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Samples (except lane1) are non-boiled as boiling may cause protein aggregates.

Exposure time: Lane 1: 10 secondsLane 2-5: 3 minutes

 

WFS1 was immunoprecipitated from 0.35 mg Mouse eyeball tissue lysate with ab259362 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259362 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse eyeball tissue lysate 40 ug

Lane 2: ab259362 IP in Mouse eyeball tissue lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab259362 in mouse eyeball tissue lysate

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

Exposure time: 50 seconds

Anti-WFS1 antibody [EPR23801-91] (ab259362) at 1/1000 dilution + Human eyeball tissue lysate at 40 µg

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 100 kDa
Observed band size: 300, 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 been described in the literature (PMID: 12913071 ).

Samples are non-boiled as boiling may cause protein aggregates. 

 Exposure time: 8 seconds

 

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling WFS1 with ab259362 at 1/5000 dilution (0.093 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on the CA1 region of mouse hippocampus (PMID: 24694561). The section was incubated with ab259362 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

All lanes : Anti-WFS1 antibody [EPR23801-91] (ab259362) at 1/1000 dilution

Lane 1 : Mouse eyeball tissue lysate at 40 µg
Lane 2 : Mouse brain tissue lysate at 40 µg
Lane 3 : Mouse spleen tissue lysate at 40 µg
Lane 4 : Rat eyeball tissue lysate at 40 µg
Lane 5 : Rat brain tissue lysate at 40 µg
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

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

Predicted band size: 100 kDa
Observed band size: 300, 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 been described in the literature (PMID: 12913071).

Low expression: spleen, PC-12 (PMID: 12913071; PMID: 11181571 ).

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time: 8 seconds

 

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labelling WFS1 with ab259362 at 1/5000 dilution (0.093 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on rat pancreatic islet. The section was incubated with ab259362 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat pancreas tissue labeling WFS1 with ab259362 at 1/50 (4.64 ug/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). Positive staining on rat pancreatic islets is observed. The nuclear counterstain was DAPI (Blue).

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).

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling WFS1 with ab259362 at 1/5000 dilution (0.093 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on the CA1 region of rat hippocampus. The section was incubated with ab259362 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling WFS1 with ab259362 at 1/5000 dilution (0.093 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control: almost no staining on mouse spleen. The section was incubated with ab259362 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins