liver FABP was immunoprecipitated using 0.5mg Mouse Liver tissue extract, 5µg of Rabbit polyclonal to liver FABP 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, Mouse Liver tissue 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 ab7847.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 13kDa; liver FABP
ICC/IF image of ab7847 stained HepG2 cells. The cells were 4% PFA 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 (ab7847, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.
All lanes : Anti-liver FABP antibody (ab7847) at 1/1000 dilutionLane 1 : Whole tissue lysate prepared from pig liverLane 2 : Whole tissue lysate prepared from pig liverLysates/proteins at 10 µg per lane.SecondaryGoat anti-rabbit HRP at 1/2000 dilutionImage courtesy of an anonymous Abreview.