重组Anti-FAK (phospho Y397)抗体[EP2160Y] - BSA and Azide free (ab271862)

Blocking buffer and concentration: 5% NFDM/TBST. 

Diluting buffer and concentration: 5% NFDM/TBST.

The activation of phosphorylation of FAK is reported to be related to developmental processes in brain tissue (PMID: 14642275, PMID: 21118706). So expression level of phosphorylated modified FAK in normal brain is quite low causing not easy to be detected. We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results.

ab181602 has been used as a loading control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab81298).

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.

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.

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.