All lanes : Anti-Niemann Pick C2 antibody [EPR19993] (ab207158) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : NPC2 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 17 kDa
Observed band size: 16-18 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab207158 observed at 16-18 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab207158 Anti-Niemann Pick C2 antibody [EPR19993] was shown to specifically react with Niemann Pick C2 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266749 (knockout cell lysate ab258079) was used. Wild-type and Niemann Pick C2 knockout samples were subjected to SDS-PAGE. ab207158 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human endometrium cancer tissue labeling Niemann Pick C2 with ab207158 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on human endometrium cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Lanes 1 & 3 : Anti-Niemann Pick C2 antibody [EPR19993] (ab207158) at 1/1000 dilution
Lane 2 : Anti-Niemann Pick C2 antibody [EPR19993] (ab207158) at 1/10000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : Human spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 17 kDa
Observed band size: 16-18 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2: 3 seconds; Lane 3: 30 seconds.

NPC2 can be glycosylated, the WB pattern is consistent with references PMID:16374838 and PMID:22183894.

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling Niemann Pick C2 with ab207158 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on human endometrium is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Niemann Pick C2 antibody [EPR19993] (ab207158) at 1/1000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
Lane 5 : Mouse spleen lysate
Lane 6 : Rat kidney lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 17 kDa
Observed band size: 16-18 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3/5: 3 minutes; Lane 4/6: 10 seconds.

NPC2 can be glycosylated, the WB pattern is consistent with references PMID:16374838 and PMID:22183894.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Niemann Pick C2 with ab207158 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic staining on mouse testis is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Niemann Pick C2 with ab207158 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.  Cytoplasmic staining on rat kidney is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.