Anti-Phosphotyrosine antibody [PY20]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Phosphotyrosine antibody [PY20] (AB10321)

ab10321 staining Phosphotyrosine in C2C12 cells treated with 2mM H2O2 for 10mins. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab10321 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• WB

Project7723****

Western blot - Anti-Phosphotyrosine antibody [PY20] (AB10321)

Cells were serum starved overnight and then incubated at room temperature for 10mins in a final concentration of 1mM sodium vanadate. PDGF was then added at a final concentration of 5ng/ml and cells were incubated at 37°C for 30mins. Vanadate inhibits endogenous phosphatases and PDGF stimulates phosphorylation. Western blots of NIH 3T3 cell lysates treated with vanadate and PDGF show an array of phosphorylated tyrosine compared to controls.

All lanes:

Western blot - Anti-Phosphotyrosine antibody [PY20] (ab10321) at 1 µg/mL

Lane 1:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 5 µg

Lane 2:

NIH 3T3 treated with Vanadate and PDGF Whole Cell Lysate at 5 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

true

Exposure time: 1min