Anti-Niemann Pick C1 antibody [EPR5209]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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 (Alexa Fluor^® 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 (Alexa Fluor^® 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.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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).

Image from Cermak S et al., PLoS One. 2016;11(11):e0167428. Fig 3.; doi: 10.1371/journal.pone.0167428. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

ab134113 staining Niemann Pick C1 in Neuro-2a (mouse neuroblastoma cell line) by intracellular 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)

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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.

Schultz 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/.

• WB

Lab

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

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.

All lanes:

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

NPC1 (Niemann Pick C1) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK293 whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Predicted band size: 142 kDa

false

• WB

Lab

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/10000 dilution

Lane 1:

HepG2 (human liver hepatocellular carcinoma cell line) cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia cell line) cell lysate at 20 µg

Lane 3:

HEK-293 (human epithelial cell line from embryonic kidney) cell lysate at 20 µg

Lane 4:

PC-3 (human prostate adenocarcinoma cell line) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 142 kDa

Observed band size: 180 kDa

false

• WB

Unknown

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

All lanes:

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/1000 dilution

Lane 1:

3T3 L1 cell lysate at 10 µg

Lane 2:

L6 (rat skeletal muscle cell line) cell lysate at 10 µg

Lane 3:

HepG2 (human liver hepatocellular carcinoma cell line) cell lysate at 10 µg

Lane 4:

THP-1 (human monocytic leukemia cell line) cell lysate at 10 µg

Lane 5:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate at 10 µg

Lane 6:

PC-3 (human prostate adenocarcinoma cell line) cell lysate at 10 µg

Lane 7:

Rat liver lysate at 10 µg

Lane 8:

Rat brain lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 142 kDa

Observed band size: 180 kDa

false

• WB

Lab

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Niemann Pick C1 antibody [EPR5209] (ab134113) at 1/10000 dilution

Lane 1:

3T3-L1 cell lysate at 20 µg

Lane 2:

L6 (rat skeletal muscle cell line) cell lysate at 20 µg

Lane 3:

Rat tissue lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 142 kDa

Observed band size: 180 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Niemann Pick C1 antibody [EPR5209] (AB134113)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.