Anti-FAK (phospho Y861) antibody

• ICC

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Immunocytochemistry - Anti-FAK (phospho Y861) antibody (AB4804)

Immunofluorescence analysis of FAK [pY861] was done on 70% confluent log phase A-549 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labelled with ab4804 at 1 : 250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labelled with a Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1 : 2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin at a 1 : 300 dilution. Panel d is a merged image showing localization of target protein at focal adhesions. Panel e is a no primary antibody control. The images were captured at 60X magnification.

• WB

AbReview20603****

Western blot - Anti-FAK (phospho Y861) antibody (AB4804)

Primary antibody incubated for 16 hours at 4°C.
Blocking step was performed using 5% milk for 1 hour at 25°C.

All lanes:

Western blot - Anti-FAK (phospho Y861) antibody (ab4804) at 1/1000 dilution

Lane 1:

Whole cell lysate prepared from human MDA-MB-231 breast cancer cells, un-treated at 25 µg

Lane 2:

Whole cell lysate prepared from human MDA-MB-231 breast cancer cells, treated for 1hr with 2.5uM AZD0530 src inhibitor at 25 µg

Lane 3:

Whole cell lysate prepared from human MDA-MB-231 breast cancer cells, treated for 1hr with 5uM AZD0530 src inhibitor at 25 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated at 1/5000 dilution

Predicted band size: 119 kDa

Observed band size: 125 kDa

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Exposure time: 5min

Image courtesy of an anonymous Abreview.

• WB

Supplier Data

Western blot - Anti-FAK (phospho Y861) antibody (AB4804)

Peptide Competition : Cell extracts prepared from chick embryo fibroblasts expressing FAK and plated on fibronectin were resolved by SDS-PAGE on a 10% Tris-glycine gel. The proteins then were transferred to nitrocellulose and incubated with 0.50 μg/mL ab4804 antibody, following prior incubation with : (1) the phosphopeptide immunogen, (2) a generic phosphotyrosine containing peptide, (3) the non-phosphorylated peptide corresponding to the phosphopeptide, and (4) no peptide. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method. The data show that only the phosphopeptide corresponding to this site blocks the antibody signal, demonstrating the specificity of the ab4804 antibody for this phosphorylated residue. Peptide Competition : Cell extracts prepared from chick embryo fibroblasts expressing FAK and plated on fibronectin were resolved by SDS-PAGE on a 10% T

All lanes:

Western blot - Anti-FAK (phospho Y861) antibody (ab4804)

Predicted band size: 119 kDa

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