Control Lysate
(HeLa cells were grown in culture plates at 37 degree C, 5% CO2 in DMEM with 10% FBS and cultured to 90% confluence. Stimulation was done with 300 nM Calyculin A for 30 min. After removal of cell culture medium, 50 uL of 1X Lysis Buffer #1 supplemented with 1 mM NaF and 2 mM Na3VO4 were added to each well and incubated for 30 min. Soluble supernatants were collected after a 10 min centrifugation and tested using the two-plate assay protocol.)
eIF2alpha assay kit
Phospho-eIF2alpha (S51) TR-FRET Cellular Assay Kit
Acc-phospho-EIF2a (20 uL)
EIF2a control lysate (100 uL)
5X Lysis Buffer #1 (1 mL)
10X TR-FRET Detection buffer (50 uL)
Control Lysate
(HeLa cells were grown in culture plates at 37 degree C, 5% CO2 in DMEM with 10% FBS and cultured to 90% confluence. Stimulation was done with 300 nM Calyculin A for 30 min. After removal of cell culture medium, 50 uL of 1X Lysis Buffer #1 supplemented with 1 mM NaF and 2 mM Na3VO4 were added to each well and incubated for 30 min. Soluble supernatants were collected after a 10 min centrifugation and tested using the two-plate assay protocol.)
Control Lysate
(HeLa cells were grown in culture plates at 37 degree C, 5% CO2 in DMEM with 10% FBS and cultured to 90% confluence. Stimulation was done with 300 nM Calyculin A for 30 min. After removal of cell culture medium, 50 uL of 1X Lysis Buffer #1 supplemented with 1 mM NaF and 2 mM Na3VO4 were added to each well and incubated for 30 min. Soluble supernatants were collected after a 10 min centrifugation and tested using the two-plate assay protocol.)
Testing Data
(HeLa cells were seeded at 20,000 cells/well and cultured overnight at 37 degree C, 5% CO2, in DMEM with 10% FBS. Cells were then treated with Calyculin A for 30 min. The media was removed from the wells, and cells were lysed with 50 uL/well of 1X Lysis Buffer #1 containing 1 mM NaF and 2 mM Na3VO4, on an orbital shaker (400 rpm) for 30 min at room temperature. The lysates were then analyzed for phospho-eIF2alpha (S51) using the two-plate assay protocol. The TR-FRET signal was recorded at 665 and 615 nm (EnVision; lamp excitation) after a 18 h incubation period at room temperature.)
Testing Data
(HeLa cells were seeded at 20,000 cells/well and cultured overnight at 37 degree C, 5% CO2, in DMEM with 10% FBS. Cells were then treated with Calyculin A for 30 min. The media was removed from the wells, and cells were lysed with 50 uL/well of 1X Lysis Buffer #1 containing 1 mM NaF and 2 mM Na3VO4, on an orbital shaker (400 rpm) for 30 min at room temperature. The lysates were then analyzed for phospho-eIF2alpha (S51) using the two-plate assay protocol. The TR-FRET signal was recorded at 665 and 615 nm (EnVision; lamp excitation) after a 18 h incubation period at room temperature.)
A superior alternative to ELISA
Features :: Benefits
• TR-FRET technology :: Proven, sensitive and robust assay technology
• Homogeneous, add-and-read assay :: No wash, no separation steps
• < 1 hour bench time :: Faster, easier than ELISA
• Adherent or suspension cells :: Higher flexibility
• Validated assays :: Results you can trust
• Small sample volume (15uL) :: Save precious samples
• Compatible with low-volume 96-well and 384-well plates : Higher throughput
• Signal stable over time :: Flexibility to read plate
• Kits available in 100 and 500 assay points :: Cost-effective and flexible
• Kits available for phospho, total and phospho+total protein :: Allows multiple protein measurements in one plate from one sample
NCBI and Uniprot Product Information
NCBI Description
The translation initiation factor EIF2 catalyzes the first regulated step of protein synthesis initiation, promoting the binding of the initiator tRNA to 40S ribosomal subunits. Binding occurs as a ternary complex of methionyl-tRNA, EIF2, and GTP. EIF2 is composed of 3 nonidentical subunits, the 36-kD EIF2-alpha subunit (EIF2S1), the 38-kD EIF2-beta subunit (EIF2S2; MIM 603908), and the 52-kD EIF2-gamma subunit (EIF2S3; MIM 300161). The rate of formation of the ternary complex is modulated by the phosphorylation state of EIF2-alpha (Ernst et al., 1987 [PubMed 2948954]).[supplied by OMIM, Feb 2010]
Uniprot Description
eIF2-alpha: a translation initiation factor that functions in the early steps of protein synthesis by forming a ternary complex with GTP and initiator tRNA. This complex binds to a 40s ribosomal subunit, followed by mRNA binding to form a 43S preinitiation complex. Junction of the 60S ribosomal subunit to form the 80S initiation complex is preceded by hydrolysis of the GTP bound to eIF-2 and release of an eIF-2-GDP binary complex. In order for eIF-2 to recycle and catalyze another round of initiation, the GDP bound to eIF-2 must exchange with GTP by way of a reaction catalyzed by eIF-2B. Phosphorylated by at least 4 kinases: PERK, GCN2, HRI and PKR. Phosphorylation stabilizes the eIF-2/GDP/eIF-2B complex and prevents GDP/GTP exchange reaction, thus impairing the recycling of eIF-2 between successive rounds of initiation and leading to global inhibition of translation. Upregulated in some thyroid cancers and bronchiolo-alveolar adenocarcinomas; aberrant phosphorylation correlates with Alzheimer disease and Epstein-Barr virus infections.
Protein type: RNA-binding; Translation; Translation initiation
Chromosomal Location of Human Ortholog: 14q23.3
Cellular Component: eukaryotic translation initiation factor 2B complex; polysome; membrane; stress granule; cytosol; nucleus; eukaryotic translation initiation factor 2 complex
Molecular Function: protein binding; translation initiation factor activity; ribosome binding
Biological Process: regulation of translation initiation in response to stress; unfolded protein response, activation of signaling protein activity; cellular protein metabolic process; translation; unfolded protein response; protein amino acid autophosphorylation; translational initiation; gene expression
Research Articles on eIF2alpha
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Product Notes
The Human, Mouse, Rat eIF2alpha eif2s1 (Catalog #AAA298016) is an Assay Kit and is intended for research purposes only. The product is available for immediate purchase. The AAA298016 Assay Kit recognizes Human, Mouse, Rat eIF2alpha. AAA Biotech's eIF2alpha can be used in a range of immunoassay formats including, but not limited to, Cell-Based Assays. Researchers should empirically determine the suitability of the eIF2alpha eif2s1 for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "eIF2alpha, Assay Kit" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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