The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/10000. Detects a band of approximately 200 kDa (predicted molecular weight: 220 kDa). EIF4G is more susceptible to degradation compared to other proteins, especially from some tissue sources such as liver. This is true even when tissue is stored at frozen. SDS-PAGE sample buffer may improve the stability, but samples that are stored frozen may show degradation bands that have been described in the literature.Therefore if multiple bands are observed in WB, it is likely due to the degradation of eIF4G rather than non-specificity of the antibody.
Use a concentration of 4 µg/ml.
Component of the protein complex eIF4F, which is involved in the recognition of the mRNA cap, ATP-dependent unwinding of 5'-terminal secondary structure and recruitment of mRNA to the ribosome.
Involvement in disease
Defects in EIF4G1 are the cause of Parkinson disease type 18 (PARK18) [MIM:614251]. An autosomal dominant, late-onset form of Parkinson disease. Parkinson disease is a complex neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity and postural instability, as well as by a clinically significant response to treatment with levodopa. The pathology involves the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (intraneuronal accumulations of aggregated proteins), in surviving neurons in various areas of the brain.
Belongs to the eIF4G family. Contains 1 MI domain. Contains 1 MIF4G domain. Contains 1 W2 domain.
Phosphorylated at multiple sites in vivo. Phosphorylation at Ser-1185 by PRKCA induces binding to MKNK1. Following infection by certain enteroviruses, rhinoviruses and aphthoviruses, EIF4G1 is cleaved by the viral protease 2A, or the leader protease in the case of aphthoviruses. This shuts down the capped cellular mRNA transcription.
ab2609 (4µg/ml) staining eIF4G1 in human colon using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of the cytoplasm of the intestinal cells. Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
ICC/IF image of ab2609 stained MCF7 cells. The cells were 4% PFA 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 (ab2609, 1µg/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.