![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.](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/330_ab108921-203451-IPVI001ab10892116m.jpg)
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 LysateLane 2 : Adrenal (Human) Whole Cell Lysate - adult normal tissue (ab29249)Lane 3 : WI38 (Human lung fibroblast cell line) 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 LysateLysates/proteins at 20 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
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.