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Synthetic peptide conjugated to KLH derived from within residues 1250 to the C-terminus of Human Niemann Pick C1.
Our Abpromise guarantee covers the use of ab108921 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use a concentration of 5 µg/ml.|
|ICC/IF||Use a concentration of 5 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 200 kDa (predicted molecular weight: 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.