CRMP5 was immunoprecipitated using 0.5mg SHSY5Y whole cell extract, 5µg of Rabbit polyclonal to CRMP5 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, SHSY5Y 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 ab79008.
Band: 70kDa; CRMP5, non specific band - 55kDa: We are unsure as to the identity of this extra band.
Western blot - CRMP5 antibody (ab79008)
All lanes : Anti-CRMP5 antibody (ab79008) at 1 µg/ml
Lane 1 : Human brain tissue lysate - total protein (ab29466) Lane 2 : Human spinal cord tissue lysate - total protein (ab29188) Lanes 3 & 5 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate Lane 4 : SK N BE (Human neuroblastoma) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
ICC/IF image of ab79008 stained SHSY5Y 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 (ab79008, 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. This antibody also gave a positive result in 100% methanol fixed (5 min) SHSY5Y cells at 5µg/ml.