All lanes : Anti-EEF2/Elongation factor 2 antibody [EP880Y] (ab75748) at 1/50000 dilution

Lane 1 : C6 (rat glioma) whole cell lysates
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysates
Lane 3 : NIH/3T3 (mouse embryo) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Rabbit monoclonal [EP880Y] to EEF2/Elongation factor 2 (ab75748) at 1/100000 dilution

Predicted band size: 95 kDa

ab75748 staining EEF2/Elongation factor 2 in rat pancreas tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/700. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

ab75748 staining EEF2/Elongation factor 2 in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody. ab7291 and ab150120 were used as counterstains for primary antibody ab75748 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

ab75748 staining EEF2/Elongation factor 2 in the human cell line HEK293 (human embryonic kidney) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/70. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

ab75748 immunoprecipitating EEF2/Elongation factor 2. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/20 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1: HeLa (human cervix adenocarcinoma) whole cell lysate (10ug)
Lane 2: HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab75748 in HeLa (human cervix adenocarcinoma) whole cell lysate

All lanes : Anti-EEF2/Elongation factor 2 antibody [EP880Y] (ab75748) at 1/50000 dilution

Lane 1 : HEK293 (human embryonic kidney) whole cell lysates
Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysates
Lane 3 : A431 (human epidermoid carcinoma) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 95 kDa
Additional bands at: 95 kDa. We are unsure as to the identity of these extra bands.

ab75748 staining EEF2/Elongation factor 2 in mouse pancreas tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody.

Negative control 1: PBS in place of primary antibody.

Overlay histogram showing HeLa cells stained with ab75748 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75748, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

EEF2/Elongation factor 2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit monoclonal to EEF2/Elongation factor 2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab75748.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 95kDa; EEF2/Elongation factor 2

ab75748 staining EEF2/Elongation factor 2 in human glioma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody.

Negative control 1: PBS in place of primary antibody.