This antibody gave a positive signal in the following whole cell lysates:
HeLa (Human epithelial carcinoma cell line)
Jurkat (Human T cell lymphoblast-like cell line)
A431 (Human epithelial carcinoma cell line)
NIH 3T3 (Mouse embryonic fibroblast cell line)
MEF1 (Mouse embryonic fibroblast cell line)
PC12 (Rat adrenal pheochromocytoma cell line)
This antibody gave a positive signal in the following tissue lysates:
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 1 µg/ml. Detects a band of approximately 47 kDa (predicted molecular weight: 47 kDa).
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml.
1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ATP-dependent RNA helicase which is a subunit of the eIF4F complex involved in cap recognition and is required for mRNA binding to ribosome. In the current model of translation initiation, eIF4A unwinds RNA secondary structures in the 5'-UTR of mRNAs which is necessary to allow efficient binding of the small ribosomal subunit, and subsequent scanning for the initiator codon.
Belongs to the DEAD box helicase family. eIF4A subfamily. Contains 1 helicase ATP-binding domain. Contains 1 helicase C-terminal domain.
ICC/IF image of ab31217 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab31217, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used to quench autofluorescence.5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
Image courtesy of Human Protein Atlas. ab31217 staining eIF4A1 in human stomach, showing strong cytoplasmic staining in the glandular cells. Paraffin embedded human stomach tissue was incubated with ab31217 (1/100 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
ab31217 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues. Further results for this antibody can be found at www.proteinatlas.org
Western blot - Anti-eIF4A1 antibody (ab31217)
All lanes : Anti-eIF4A1 antibody (ab31217) 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 : Liver (Mouse) Tissue Lysate - normal tissue Lane 4 :Mouse pancreas tissue lysate - total protein (ab29363) Lane 5 : Testis (Mouse) Tissue Lysate - normal tissue Lane 6 : PC12 (Rat adrenal pheochromocytoma 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 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 47 kDa Observed band size: 47 kDa
eIF4A1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to eIF4A1 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 ab31217. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 47kDa: eIF4A1
Amorim IS et al. Loss of eIF4E Phosphorylation Engenders Depression-like Behaviors via Selective mRNA Translation. J Neurosci38:2118-2133 (2018).
Read more (PubMed: 29367404) »
Passacantilli I et al. Alternative polyadenylation of ZEB1 promotes its translation during genotoxic stress in pancreatic cancer cells. Cell Death Dis8:e3168 (2017).
Read more (PubMed: 29120411) »