All lanes : Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : NPC1 (Niemann Pick C1) knockout HAP1 whole cell lysate
Lane 3 : HEK293 whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 142 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab134113 observed at 180 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab134113 was shown to specifically react with Niemann Pick C1 in wild-type HAP1 cells as signal was lost in NPC1 (Niemann Pick C1) knockout cells. Wild-type and NPC1 (Niemann Pick C1) knockout samples were subjected to SDS-PAGE. Ab134113 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma cell line) cells labeling Niemann Pick C1 with ab134113 at 1/70 followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500(green). Confocal image showing cytoplasmic staining on Neuro-2a cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 and ab150120(AlexaFluor®594 Goat anti-Mouse secondary) at 1/500  (red).

The negative controls are as follows:
-ve control 1 – ab134113 at 1/70 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500.

Immunohistochemical staining of paraffin embedded human liver with purified ab134113 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

ab134113 staining Niemann Pick C1 in Neuro-2a (mouse neuroblastoma cell line) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/200. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabeled control: Cell without incubation with primary antibody and secondary antibody (Blue)

I1061T NPC1 traffics to autophagosomes 

I1061T NPC1 fibroblasts were treated with vehicle or 100 nM bafilomycin A1 (Baf) for 24 h. Fixed cells were stained for LC3 (red), NPC1 (green), and DAPI (blue) then imaged by confocal microscopy. Co-localization is indicated by yellow color in the merged image. Scale bar = 25 μm.

Niemann Pick C1 (NPC1) was detected using ab134113 at 1/200 dilution.

From Figure 4b of Schultz et al.

Shultz et al Nat Commun. 2018; 9: 3671.Published online 2018 Sep 10. doi: 10.1038/s41467-018-06115-2

Reproduced under the Creative Commons Licence http://creativecommons.org/licenses/by/4.0/.

Cathepsin B/L inhibition causes NPC disease-like cholesterol accumulation in SH-SY5Y cells.

Confocal microscopy of SH-SY5Y control and PADK treated cells. Cholesterol (filipin staining, white) and NPC1 (ab134113; green).

All lanes : Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/10000 dilution (purified)

Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate
Lane 2 : THP-1 (human monocytic leukemia cell line) cell lysate
Lane 3 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate
Lane 4 : PC-3 (human prostate adenocarcinoma cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 142 kDa
Additional bands at: 180 kDa (possible glycosylated form)

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/10000 dilution (purified)

Lane 1 : 3T3-L1 cell lysate
Lane 2 : L6 (rat skeletal muscle cell line) cell lysate
Lane 3 : Rat tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 142 kDa
Additional bands at: 180 kDa (possible glycosylated form)

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded mouse liver with purified ab134113 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded rat cerebellum with purified ab134113 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

All lanes : Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/1000 dilution (unpurified)

Lane 1 : 3T3 L1 cell lysate
Lane 2 : L6 (rat skeletal muscle cell line) cell lysate
Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) cell lysate
Lane 4 : THP-1 (human monocytic leukemia cell line) cell lysate
Lane 5 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
Lane 6 : PC-3 (human prostate adenocarcinoma cell line) cell lysate
Lane 7 : Rat liver lysate
Lane 8 : Rat brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 142 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin embedded human kidney tissue labelling Niemann Pick C1 with unpurified ab134113 at 1/50.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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