Anti-Ret (phospho Y1062) antibody [EPR30639-507]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized TT (human thyroid carcinoma epithelial cell) untreated and TT cell treated with 100 µM pervanadate for 10 mins labelling Ret with ab326572 at 1/50 (9.94 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased membranous and cytoplasmic staining in TT cells (shown in green) treated with 100 µM pervanadate for 10 mins. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IP

Lab

Immunoprecipitation - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

Ret was immunoprecipitated from 0.35 mg TT (human thyroid carcinoma epithelial cell) treated with mM pervanadate for 30 min whole cell lysate with ab326572 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326572 at 1/326572 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : TT (human thyroid carcinoma epithelial cell) treated with 1mM pervanadate for 30 min whole cell lysate
Lane 2 : ab326572 IP in TT (human thyroid carcinoma epithelial cell) treated with mM pervanadate for 30 min whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326572 in TT (human thyroid carcinoma epithelial cell) treated with 1mM pervanadate for 30 min whole cell lysate

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/5000 dilution

Lane 1:

TT (human thyroid carcinoma epithelial cell) treated with 1mM pervanadate for 30 min whole cell lysate at 10 µg

Lane 2:

ab326572 at 1/30 IP in TT (human thyroid carcinoma epithelial cell) treated with mM pervanadate for 30 min whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab326572 in TT (human thyroid carcinoma epithelial cell) treated with 1mM pervanadate for 30 min whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 150 kDa

false

Exposure time: 10s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) untreated and Neuron-2a cell treated with 100 µM pervanadate for 10 mins labelling Ret with ab326572 at 1/50 (9.94 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased membranous and cytoplasmic staining in Neuron-2a cells (shown in green) treated with 100 µM pervanadate for 10 mins. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IP

Lab

Immunoprecipitation - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

Ret was immunoprecipitated from 0.35 mg Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate with ab326572 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab326572 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate
Lane 2 : ab326572 IP in Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab326572 in Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate

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

Exposure time : 75 seconds.

All lanes:

Immunoprecipitation - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/1000 dilution

Lane 1:

Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate at 10 µg

Lane 2:

ab326572 at 1/30 IP in Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab326572 in Neuro-2a (mouse neuroblastoma neuroblast) treated with 100µM pervanadate for 10 min whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 150 kDa

false

Exposure time: 75s

• WB

Lab

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

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

The antibody shows weak reactivity with HEK293T cell lines overexpressing human Ret (Y1062A mutant)

In Western blot, Anti-Ret antibody (ab134100) (1 : 1000) (150KDa); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(150KDa); Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda).

All lanes:

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a his-tag treated with 100uM pervanadate for 10 min whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human wild-type Ret expression vector containing a his-tag were treated with 100uM Pervanadate for 10 mins, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human Ret(Y1062A mutant) expression vector containing a his-tag were treated with 100uM Pervanadate for 10 mins, whole cell lysate at 2 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 1/100000 dilution

Observed band size: 150 kDa,15 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa);Anti-Ret antibody (ab134100) (1 : 1000) (150KDa).

All lanes:

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/1000 dilution

Lane 1:

Untreated TT (human thyroid carcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

TT treated with 100uM pervanadate for 10 min whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated TT (human thyroid carcinoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 4:

TT treated with 100uM pervanadate for 10 min whole cell lysate (alkaline phosphatase treated membrane) at 20 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: 150 kDa,36 kDa

false

Exposure time: 6s

• Dot

Lab

Dot Blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

Dot blot analysis of Ret using ab326572 at 1 : 1000 (0.497 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : Human Ret (phospho Y1062) peptide a

Lane 2 : Human Ret (phospho Y1062) peptide b

Lane 3 : Human Ret non-phospho peptide

Exposure time : 180 seconds.

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

All lanes:

Dot Blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/1000 dilution

Lane 1:

Human Ret (phospho Y1062) peptide a

Lane 2:

Human Ret (phospho Y1062) peptide b

Lane 3:

Human Ret non-phospho 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

• WB

Lab

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (AB326572)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa);Anti-Ret antibody (ab134100) (1 : 1000) (150KDa).

All lanes:

Western blot - Anti-Ret (phospho Y1062) antibody [EPR30639-507] (ab326572) at 1/1000 dilution

Lane 1:

Neuro-2a treated with 100uM pervanadate for 10 min whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Untreated Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

Lane 4:

Neuro-2a treated with 100uM pervanadate for 10 min whole cell lysate (alkaline phosphatase treated membrane) at 20 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: 150 kDa,36 kDa

false

Exposure time: 37s