Anti-Phosphotyrosine antibody [EPR16871]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed Pervanadate (50mM, 30min.) treated (orange)/untreated (red)A431 (Human epidermoid carcinoma) cells labeling Phosphotyrosine with ab179530 at 1/160 dilutioncompared with a rabbit monoclonal IgG control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C2C12 (Mouse myoblast cell line) cells labeling Phosphotyrosine with ab179530 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Cytoplasm staining on C2C12 cells is observed. The expression increased after treatment with H2O2 (2mM) for 10 minutes. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows : -
-ve control 1 : - ab179530 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2 : - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

• WB

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Western blot - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Multiple bands represent phosph-tyrosine containing proteins detected by ab179530.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (ab179530) at 1/10000 dilution

Lane 1:

A431 (Human epidermoid carcinoma) whole cell lysates treated with 50mM pervanadate for 30 minutes at 10 µg

Lane 2:

Untreated A431 whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

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• WB

Supplier Data

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Multiple bands represent phosph-tyrosine containing proteins detected by ab179530

All lanes:

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (ab179530) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates treated with 1mM pervanadate for 20minutes at 10 µg

Lane 2:

Untreated Jurkat whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (H+L) Peroxidase conjugated at 1/1000 dilution

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• IP

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Immunoprecipitation - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Phosphotyrosine was immunoprecipitated from 1mg of A431 (Human epidermoid carcinoma) whole cell extract treated with 1mM pervanadate for 30 minutes with ab179530 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab179530 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : A431 treated with 1mM pervanadate for 30 minutes whole cell extract. Lane 2 : PBS instead of A431 whole cell extract.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Multiple bands represent phosph-tyrosine containing proteins precipitated and detected by ab179530.

All lanes:

Immunoprecipitation - Anti-Phosphotyrosine antibody [EPR16871] (ab179530)

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• ELISA

Lab

ELISA - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Serially diluted ab179530 was bound to immobilised phospho- or control peptides (STAT1 (phospho S727), STAT1 control, STAT5 (phospho T694), STAT5 control); 1 microgram per mL).

The antibody was detected by Goat anti-Rabbit HRPO and signal was developed by TMB substrate.

• ELISA

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ELISA - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

ELISA analysis of various antigens (1 μg/ml) using ab179530 at 1/6400 dilution followed by Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution.

S/N = signal-to-noise ratio of phospho- versus nonphospho-peptides.

• WB

Supplier Data

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the bands around 50 kDa are unknown. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-phosphotyrosine antibody [EPR16871] (ab179530) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-3-Nitrotyrosine antibody [EPR27217-88] (<a href='/en-us/products/primary-antibodies/3-nitrotyrosine-antibody-epr27217-88-ab314438'>ab314438</a>) at 1/1000 dilution

Lanes 1 and 7:

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

Lane 2:

HeLa treated with 3 mM peroxynitrite for 30 minutes whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

NIH/3T3 treated with 3 mM peroxynitrite for 30 minutes whole cell lysate at 20 µg

Lane 5:

Untreated PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 6:

PC-12 treated with 3 mM peroxynitrite for 30 minutes whole cell lysate at 20 µg

Lane 8:

HeLa treated with 1 mM pervanadate for 30 minutes whole cell 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 1/100000 dilution

Observed band size: 1-250 kDa

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Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Multiple bands represent phosph-tyrosine containing proteins detected by ab179530.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (ab179530) at 1/1000 dilution

Lane 1:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates treated with 1mM pervanadate for 10 minutes at 10 µg

Lane 2:

Untreated NIH/3T3 whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

false

• WB

Supplier Data

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Multiple bands represent phosph-tyrosine containing proteins detected by ab179530.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Phosphotyrosine antibody [EPR16871] (ab179530) at 1/1000 dilution

Lane 1:

L6 (Rat skeletal muscle cell line) whole cell lysates treated with 1mM pervanadate for 20 minutes at 10 µg

Lane 2:

Untreated L6 whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

false

• Dot

Lab

Dot Blot - Anti-Phosphotyrosine antibody [EPR16871] (AB179530)

Dot blot analysis of INSR/IGF-1R (pY1009) phospho peptide (lane 1) and INSR/IGF-1R non-phospho peptide (lane 2) labelling Phosphotyrosine with ab179530 at a dilution of 1/1000. A peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/2500).

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.