Lane 1 : Marker
Lanes 2-4 : Anti-beta Arrestin 1 antibody (ab31868) at 1 µg/ml

Lane 2 : Mouse Brain at 20 µg
Lane 3 : Mouse brain tissue lysate - total protein (0 days) (ab7188) at 20 µg
Lane 4 : Brain (Rat) Whole Cell Lysate - normal tissue at 20 µg

Secondary
Lanes 2-4 : IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 50 kDa

why is the actual band size different from the predicted?
Additional bands at: 100 kDa. We are unsure as to the identity of these extra bands.

ICC/IF image of ab31868 stained PC12 cells. The cells were 100% methanol fixed (5 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 (ab31868, 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.

IHC image of ab31868 staining in rat brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab31868, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Beta Arrestin 1 was immunoprecipitated using 0.5mg Mouse Brain tissue lysate, 5µg of Rabbit polyclonal to Beta Arrestin 1 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 Brain tissue lysate 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31868.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 50kDa: Beta Arrestin 1