The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Detects a band of approximately 47 kDa (predicted molecular weight: 47 kDa).
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.
Anti-eIF4A2 antibody (ab31218) at 1 µg/ml + Jurkat whole cell lysate (ab7899) at 20 µg
Secondary IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution
Performed under reducing conditions.
Predicted band size: 47 kDa Observed band size: 47 kDa Additional bands at: 52 kDa. We are unsure as to the identity of these extra bands.
Immunocytochemistry/ Immunofluorescence - Anti-eIF4A2 antibody (ab31218)This image is courtesy of an anonymous Abreview
Immunocytochemistry/ Immunofluorescence analysis of MDA-MB231 Cells labeling eIF4A2 with ab31218 at a dilution of 1/200. Cells were fixed with Paraformaldehyde and permeabilized with 0.2% Saponin. The blocking step was incubation with 5% BSA for 30 minutes at 22°C. An Alexa Flour® 488 conjugated donkey anti-rabbit was used as the secondary antibody.
Image courtesy of Human Protein Atlas. ab31218 staining eIF4A2 in human duodenum, showing strong cytomplasmic staining in the villi and Brunner's gland cells. Paraffin embedded human duodenum tissue was incubated with ab31218 (1/400 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
ab31218 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
eIF4A2 was immunoprecipitated using 0.5mg Mouse skeletal muscle whole tissue extract, 5µg of Rabbit polyclonal to eIF4A2 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, Mouse skeletal muscle whole tissue 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 ab31218. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 47kDa: eIF4A2 .
Western blot - Anti-eIF4A2 antibody (ab31218)
All lanes : Anti-eIF4A2 antibody (ab31218) at 1 µg/ml
Lane 1 : Brain (Mouse) Tissue Lysate Lane 2 : Testis (Mouse) Tissue Lysate Lane 3 :Mouse skeletal muscle tissue lysate - total protein (ab29711) Lane 4 : Spinal Cord (Mouse) Tissue Lysate Lane 5 : Ovary (Mouse) Tissue Lysate Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate Lane 7 : Brain (Rat) Tissue Lysate Lane 8 : Heart (Rat) Tissue 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 Additional bands at: 51 kDa. We are unsure as to the identity of these extra bands.