Western blot - VASP antibody (ab26650)

All lanes : Anti-VASP antibody (ab26650) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate (ab7909)

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 40 kDa
Observed band size : 43 kDa (why is the actual band size different from the predicted?)
Additional bands at : 49 kDa (possible cross reactivity).

Western blot - VASP antibody (ab26650)

All lanes : Anti-VASP antibody (ab26650) at 1 µg/ml

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 40 kDa
Observed band size : 43 kDa (why is the actual band size different from the predicted?)

Immunocytochemistry/ Immunofluorescence - VASP antibody (ab26650)

ICC/IF image of ab26650 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab26650, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

Western blot - VASP antibody (ab26650)

All lanes : Anti-VASP antibody (ab26650) at 1 µg/ml

Lane 1 : HUVEC (Human Umbilical Vein Endothelial Cell) Whole Cell Lysate
Lane 2 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 40 kDa
Observed band size : 45,49 kDa (why is the actual band size different from the predicted?)
Additional bands at : 86 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 20 minutes

Flow Cytometry - VASP antibody (ab26650)

ab26650 detecting VASP in Human platelets by Flow Cytometry. Platelets were isolated spinning Platelet rich plasma on Histopaque. Cells were fixed in PFA and permeabilized in 0.1% Triton-X100 in 2% BSA for 30 minutes. The primary antibody was diluted 1/200 in 2% BSA in PBS and incubated with the sample for 18 hours at 4ºC. An Alexa Fluor® 488-conjugated Chicken anti-Rabbit IgG (H+L) antibody, diluted 1/500 was used as the secondary antibody.

Abbreviations in the figure:
Plts: Platelets;
UP: Unpermeabilized;
US Unstained, Red Peak;
IgG Rb: IgG Rabbit (Isotype Control), Blue Peak;
VASP: VASP, Green peak.

This image is courtesy of Mahesh Shivananjappa.