Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133]

• WB

Supplier Data

Western blot - Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133] (AB324617)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

U-118MG cells carry the FIG-ROS1 fusion (PMID : 12661006). Tyrosine phosphorylated ROS1 is downregulated in response to crizotinib treatment (PMID : 24971615)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133] (ab324617) at 1/1000 dilution

Lane 1:

Untreated U-118 MG (human brain glioblastoma cell) whole cell lysate (untreated membrane) at 50 µg

Lane 2:

U-118 MG treated with 250 nM crizotinib for 4 hours whole cell lysate (untreated membrane) at 50 µg

Lane 3:

Untreated U-118 MG whole cell lysate (alkaline phosphatase treated membrane) at 50 µg

Lane 4:

U-118 MG treated with 250 nM crizotinib for 4 hours whole cell lysate (alkaline phosphatase treated membrane) at 50 µg

Secondary

All lanes:

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

Observed band size: 110 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133] (AB324617)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Tyrosine phosphorylated ROS1 is high in ROS1(D2113N mutant) overexpressing cells (PMID : 37587872) when compared to wildtype ROS1. The signal is reduced in cells overexpressing ROS1(D2113N, Y2274F mutant) and absent in cells overexpressing ROS1(Y2274F, Y2334F mutant).

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133] (ab324617) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with a human ROS1 expression vector containing a myc-His-tag®, whole cell lysate at 50 µg

Lane 2:

293T cells transfected with a human ROS1(D2113N mutant) expression vector containing a myc-His-tag®, whole cell lysate at 50 µg

Lane 3:

293T cells transfected with a human ROS1(D2113N, Y2274F mutant) expression vector containing a myc-His-tag®, whole cell lysate at 50 µg

Lane 4:

293T cells transfected with a human ROS1(Y2274F, Y2334F mutant) expression vector containing a myc-His-tag®, whole cell lysate at 50 µg

Secondary

All lanes:

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

Observed band size: 265 kDa

false

Exposure time: 92s

• Dot

Supplier Data

Dot Blot - Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133] (AB324617)

Dot blot analysis of Phospho-ROS1 (Tyr2274/2334) using ab324617 at 1 : 1000 (0.489 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane1 : ROS1 (phospho Y2274) peptide
Lane2 : ROS1 non-phospho peptide
Lane3 : ROS1 (phospho Y2334) peptide

Exposure time : 180 seconds.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The antibody recognizes ROS1 protein when singly phosphorylated at Tyr2274, and shows slight reactivity with ROS1 when singly phosphorylated at Tyr2334.

All lanes:

Dot Blot - Anti-Phospho-ROS1 (Tyr2274/2334) antibody [EPR28163-133] (ab324617) at 1/1000 dilution

Lane 1:

ROS1 (phospho Y2274) peptide

Lane 2:

ROS1 non-phospho peptide

Lane 3:

ROS1 (phospho Y2334) peptide

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s