Anti-FAK (phospho Y397) antibody [EP2160Y] - Low endotoxin, Azide free

• WB

Supplier Data

Western blot - Anti-FAK (phospho Y397) antibody [EP2160Y] - Low endotoxin, Azide free (AB223529)

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).

Lane 1:

Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lanes 1 - 5:

Western blot - Anti-FAK (phospho Y397) antibody [EP2160Y] (<a href='/en-us/products/primary-antibodies/fak-phospho-y397-antibody-ep2160y-ab81298'>ab81298</a>) at 1/1000 dilution

Lane 2:

NIH/3T3 treated with 10mM Pervanadate for 60 min whole cell lysate at 20 µg

Lane 3:

Untreated HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa treated with 10mM Pervanadate for 60 min whole cell lysate at 20 µg

Lane 5:

Mouse brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 119 kDa

Observed band size: 119 kDa

false

Exposure time: 40s

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-FAK (phospho Y397) antibody [EP2160Y] - Low endotoxin, Azide free (AB223529)

This ICC/IF data was generated using the same anti-FAK (phospho Y397) antibody clone [EP2160Y] in a different buffer formulation (cat# ab81298).

ICC/IF image of unpurified ab81298 stained SKNSH 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-FAK (phospho Y397) antibody [EP2160Y] - Low endotoxin, Azide free (AB223529)

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.

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