ab33523 staining EEF2 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 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 ab33523 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

All lanes : Anti-EEF2/Elongation factor 2 antibody (ab33523) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat whole cell lysate (ab7899)
Lane 3 : A-431 whole cell lysate (ab7909)
Lane 4 : HEK-293 whole cell lysate (ab7902)
Lane 5 : Hep G2 whole cell lysate (ab7900)
Lane 6 : MCF-7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 7 : SHSY-5Y (Human Neuroblastoma) Whole Cell Lysate
Lane 8 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size: 95 kDa
Observed band size: 95 kDa

All lanes : Anti-EEF2/Elongation factor 2 antibody (ab33523) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Mouse pancreas tissue lysate - total protein (ab29363)
Lane 4 : Ovary (Mouse) Tissue Lysate - normal tissue
Lane 5 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 6 : Liver (Rat) Tissue Lysate - normal tissue

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 95 kDa
Observed band size: 95 kDa

All lanes : Anti-EEF2/Elongation factor 2 antibody (ab33523) at 1 µg/ml

Lane 1 : Marker
Lane 2 : Zebrafish brain homogenate (20ug)
Lane 3 : Zebrafish heart homogenate (20ug)
Lane 4 : Zebrafish liver homogenate (20ug)
Lane 5 : Zebrafish skeletal muscle homogenate (20ug)
Lane 6 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (20ug)

Secondary
All lanes : Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 95 kDa
Observed band size: 95 kDa


Exposure time: 5 minutes

IHC image of EEF2/Elongation factor 2 staining in human lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab33523, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

EEF2/Elongation factor 2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal 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 ab33523.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 95kDa; EEF2/Elongation factor 2, non-specific band: 135kDa,due to background seen in No ab control lane (2).