PDIA6 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to PDIA6 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 ab11432.
ICC/IF image of ab11432 stained HeLa 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 (ab11432, 1µg/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.
Groenendyk J et al. Interplay between the oxidoreductase PDIA6 and microRNA-322 controls the response to disrupted endoplasmic reticulum calcium homeostasis. Sci Signal7:ra54 (2014).
Read more (PubMed: 24917591) »
Wang X et al. An six-amino acid motif in the alpha3 domain of MICA is the cancer therapeutic target to inhibit shedding. Biochem Biophys Res Commun387:476-81 (2009).
Read more (PubMed: 19615970) »