Immunocytochemistry/Immunofluorescence analysis of HeLa cells (untreated and treated with 1mM pervanadate for 15 minutes) labelling SRC Family (phospho Y418) with purified ab40660 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Flow Cytometry analysis of HeLa cells (treated with 1mM pervanadate for 30 minutes) labelling SRC Family (phospho Y418) with purified ab40660 at 1/80 (red). Cells were fixed with 4% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody.

Black - Isotype control, rabbit monoclonal IgG with pervanade treated HeLa cells.

Blue - Unlabelled control, pervanadate treated HeLa cells without incubation with primary and secondary antibodies.

Red - Pervandate treated HeLa cells with ab40660.

Green - Untreated HeLa cells labelled with ab40660.

All lanes : Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660) at 1/2000 dilution (purified)

Lane 1 : Untreated HeLa whole cell lysate
Lane 2 : Pervanadate treated HeLa whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 61 kDa
Observed band size: 56-59 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking and dilution buffer: 5% BSA/TBST.

All lanes : Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660) at 1/1000 dilution (purified)

Lane 1 : Pervanadate treated Jurkat cell lysate with Lyn non-phospho peptide
Lane 2 : Pervanadate treated Jurkat cell lysate with Lyn (pY396) phospho peptide
Lane 3 : Pervanadate treated Jurkat cell lysate with Lck non-phospho peptide
Lane 4 : Pervanadate treated Jurkat cell lysate with Lck (pY393) phospho peptide
Lane 5 : Pervanadate treated Jurkat cell lysate with Src non-phospho peptide
Lane 6 : Pervanadate treated Jurkat cell lysate with Src (pY418) phospho peptide

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 61 kDa
Observed band size: 56-59 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660) at 1/1000 dilution (purified)

Lane 1 : Untreated C6 whole cell lysate
Lane 2 : Pervanadate treated C6 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 61 kDa
Observed band size: 56-59 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

Blocking and dilution buffer: 5% BSA/TBST.

ICC/IF image of unpurified ab40660 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab40660, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Direct ELISA antibody dose-response curve using unpurified ab40660 at 0-1000 ng/ml. Antigen concentration of 1000 ng/mL. An alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.