This antibody gave a positive signal in the following whole cell lysates:
HeLa; Jurkat; A431; HEK 293; HepG2; MCF-7; SHSY-5Y; U2OS; NIH 3T3; MEF1; PC12.
This antibody gave a positive signal in the following tissue lysates:
Mouse Pancreas; Mouse Ovary; Rat Liver.
IHC-P: human lung FFPE tissue sections
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration. Detects a band of approximately 95 kDa (predicted molecular weight: 95 kDa).Can be blocked with Human EEF2 peptide (ab33522).
Catalyzes the GTP-dependent ribosomal translocation step during translation elongation. During this step, the ribosome changes from the pre-translocational (PRE) to the post-translocational (POST) state as the newly formed A-site-bound peptidyl-tRNA and P-site-bound deacylated tRNA move to the P and E sites, respectively. Catalyzes the coordinated movement of the two tRNA molecules, the mRNA and conformational changes in the ribosome.
Belongs to the GTP-binding elongation factor family. EF-G/EF-2 subfamily.
Phosphorylation by EF-2 kinase completely inactivates EF-2. Diphthamide is 2-[3-carboxyamido-3-(trimethyl-ammonio)propyl]histidine. Diphthamide can be ADP-ribosylated by diphtheria toxin and by Pseudomonas exotoxin A, thus arresting protein synthesis. ISGylated.
ICC/IF image of ab33523 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab33523, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal 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 - Anti-EEF2 antibody (ab33523)
All lanes : Anti-EEF2 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
IHC image of EEF2 staining in human lung formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to EEF2 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 70oC; 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, non-specific band: 135kDa,due to background seen in No ab control lane (2).
Kissing S et al. Disruption of the vacuolar-type H+-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes. Autophagy13:670-685 (2017).
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Sawhney S et al. Alpha-enolase is upregulated on the cell surface and responds to plasminogen activation in mice expressing a ?133p53a mimic. PLoS One10:e0116270 (2015).
Read more (PubMed: 25643152) »