Figure 2. The relationship between the protein concentration of lysates from Jurkat cells treated with calyculin A and pervanadate or LY294002 and the absorbance at 450 nm using PathScan® Total eIF2α Sandwich ELISA Kit is shown.
Figure 1. Treatment of Jurkat cells with 100 nM Calyculin A #9902 and 1 mM pervanadate increases phosphorylation of eIF2α at Ser51, detected by the PathScan® Phospho-eIF2α (Ser51) Sandwich ELISA Kit #7948, but does not affect the levels of total eIF2α detected by PathScan® Total eIF2α Sandwich ELISA Kit #7952. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using eIF2α (L57A5) Mouse mAb #2103 (left panel) or Phospho-eIF2α (Ser51) (119A11) Rabbit mAb #3597 (right panel) are shown in the bottom figure.
|Product Includes||Volume||Solution Color|
|eIF2α Mouse mAb Coated Microwells||96 tests|
|eIF2-α Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total eIF2α Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of eIF2α protein. A eIF2α rabbit antibody has been coated onto the microwells. After incubation with cell lysates, eIF2α (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a eIF2α mouse antibody is added to detect captured eIF2α protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate (TMB) is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of total eIF2α protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total eIF2α Sandwich ELISA Kit #7952 detects endogenous levels of total eIF2α protein in human cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Phosphorylation of the eukaryotic initiation factor 2 (eIF2) α subunit is a well-documented mechanism to downregulate protein synthesis under a variety of stress conditions. eIF2 binds GTP and Met-tRNAi and transfers Met-tRNA to the 40S subunit to form the 43S preinitiation complex (1,2). eIF2 promotes a new round of translation initiation by exchanging GDP for GTP, a reaction catalyzed by eIF2B (1,2). Kinases that are activated by viral infection (PKR), endoplasmic reticulum stress (PERK/PEK), amino acid deprivation (GCN2), or heme deficiency (HRI) can phosphorylate the α subunit of eIF2 (3,4). This phosphorylation stabilizes the eIF2-GDP-eIF2B complex and inhibits the turnover of eIF2B. Induction of PKR by IFN-γ and TNF-α induces potent phosphorylation of eIF2α at Ser51 (5,6).
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