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.

 

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.

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.

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 : 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
Lane 2 : Untreated Jurkat whole cell lysates

Lysates/proteins at 10 µg per lane.

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

Multiple bands represent phosph-tyrosine containing proteins detected by ab179530

All lanes : 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
Lane 2 : Untreated A431 whole cell lysates

Lysates/proteins at 10 µg per lane.

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

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

Blocking/Dilution buffer: 5% NFDM/TBST.

 

All lanes : 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
Lane 2 : Untreated NIH/3T3 whole cell lysates

Lysates/proteins at 10 µg per lane.

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

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

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : 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
Lane 2 : Untreated L6 whole cell lysates

Lysates/proteins at 10 µg per lane.

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

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

Blocking/Dilution buffer: 5% NFDM/TBST.

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 dilution compared 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.

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.