All lanes : Anti-Niemann Pick C1 antibody (ab108921) at 1 µg/ml

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 - ab108921 observed at 200 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab108921 was shown to recognize Niemann Pick C1 in wild-type HAP1 cells as signal was lost at the expected MW in NPC1 (Niemann Pick C1) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NPC1 (Niemann Pick C1) knockout samples were subjected to SDS-PAGE. Ab108921 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml 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.

Niemann Pick C1 was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to Niemann Pick C1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab108921.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 142kDa; Niemann Pick C1, non specific bands - 55kDa: We are unsure as to the identity of this extra band.

All lanes : Anti-Niemann Pick C1 antibody (ab108921) at 1 µg/ml (Milk Blocking - 3%)

Lane 1 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 2 : Human adrenal normal tissue lysate - total protein (ab29249)
Lane 3 : WI-38 whole cell lysate (ab3960)
Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 5 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 142 kDa
Observed band size: 200 kDa

why is the actual band size different from the predicted?
Additional bands at: 42 kDa, 60 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes


Niemann Pick C1 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

ICC/IF image of ab108921 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab108921, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) HeLa cells at 5µg/ml, and in 100% methanol fixed (5 min) HepG2 cells at 5µg/ml.