All lanes : Anti-FAK (phospho Y397) antibody [EP2160Y] (ab81298) at 1/2000 dilution (purified)

Lane 1 : Untreated mouse brain
Lane 2 : Mouse brain treated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 119 kDa
Observed band size: 119 kDa

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

ICC/IF image of unpurified ab81298 stained SK-N-SH (human neuroblastoma cell line) 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 (ab81298, 10µg/ml) overnight at +4°C in PBS containing 1% BSA and 0.1% tween. 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.

All lanes : Anti-FAK (phospho Y397) antibody [EP2160Y] (ab81298) at 1/1000 dilution (purified)

Lane 1 : Untreated rat brain
Lane 2 : Rat brain treated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP conjugated goat anti-rabbit IgG (H+L) at 1000 µg

Predicted band size: 119 kDa
Observed band size: 119 kDa

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Unpurified ab81298 staining FAK (phospho Y397) in SK-N-SH (human neuroblastoma cell line) cells treated with anandamide (in water soluble emulsion) (ab120429), by ICC/IF. Increase in FAK (phospho Y397) expression correlates with increased concentration of anandamide (in water soluble emulsion), as described in literature.
The cells were incubated at 37°C for 5 minutes in media containing different concentrations of ab120429 (anandamide (in water soluble emulsion)) in water, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab81298 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue. Membranes are stained in red with WGA.

All lanes : Anti-FAK (phospho Y397) antibody [EP2160Y] (ab81298) at 1/2000 dilution (unpurified)

Lane 1 : human brain tissue lysates, untreated.
Lane 2 : human brain tissue lysates treated with AP.
Lane 3 : rat brain tissue lysates, untreated.
Lane 4 : rat brain tissue lysates treated with AP.

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti rabbit. at 1/2000 dilution

Predicted band size: 119 kDa
Observed band size: 119 kDa

Unpurified ab81298 staining FAK (phospho Y397) in SK-N-SH (human neuroblastoma cell line) cells treated with anandamide (ethanol solution) (ab120087), by ICC/IF. Increase in FAK (phospho Y397) expression correlates with increased concentration of anandamide (ethanol solution), as described in literature.
The cells were incubated at 37°C for 10 minutes in media containing different concentrations of ab120087 (anandamide (ethanol solution)) in ethanol, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab81298 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue. Membranes are stained in red with WGA.