Methylglyoxal (MGO), TUDCA, wortmannin, U73122, compound C, and SP600125 were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) reagents and apelin-13 were obtained from Amresco (Solon, OH, USA) and American Peptide (Sunnyvale, CA, USA), respectively. Antibodies were purchased from the following vendors: KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany); ATF4, GADD153 (CHOP), PERK, phospho-PERK, eIF2α, and α-actin (Santa Cruz, CA, USA); PARP-1, cleaved caspase-3, VE-cadherin, phospho-elF2α, JNK, and phospho-JNK (Cell Signaling Technology, Danvers, MA, USA); and α-tubulin (Sigma, St. Louis, MO, USA).
Phospho elf2α
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-eIF2α is a laboratory reagent used to detect and quantify the phosphorylation of the eIF2α protein. eIF2α is a critical component of the eukaryotic translation initiation complex, and its phosphorylation is a key regulatory mechanism in the cellular stress response. This product can be used to measure the level of phosphorylated eIF2α in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Parp1, by Cell Signaling Technology (3 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
Elf2α, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
U73122, by Merck Group (1 mentions)
α actin, by Santa Cruz Biotechnology (1 mentions)
Wortmannin, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Tudca, by Merck Group (1 mentions)
Phospho perk, by Santa Cruz Biotechnology (1 mentions)
Methylglyoxal mgo, by Merck Group (1 mentions)
Eif2α, by Santa Cruz Biotechnology (1 mentions)
Sp600125, by Merck Group (1 mentions)
Ve cadherin, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Compound c, by Merck Group (1 mentions)
Anti lc3, by Novus Biologicals (1 mentions)
Iblot dry transfer system, by Thermo Fisher Scientific (1 mentions)
Parp1, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
4 protocols using phospho elf2α
1
Endoplasmic Reticulum Stress Pathway Evaluation
2
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were prepared either in 20 mM Tris pH 7.9, 120 mM KCl, 5 mM MgCl2 0.2% Nonidet P-40, 5 mM EDTA, 10% glycerol (for PARP1 and Casp3 analysis) or in 100 mM Tris pH 8, 140 mM NaCl, 20 mM EDTA, 0.2% SDS, 1% Nonidet P-40 lysis buffer, resolved on SDS-PAGE gels and transferred onto PVDF membranes using the iBlot dry Transfer System (Life Technologies, Carlsbad, CA, USA). Blocked membranes were incubated with the following primary antibodies: Atf-4 (1 : 500); elF2α (1 : 1000); phospho-elF2α (1 : 500); PARP1 (1 : 1000, Cell Signaling Technology, CST, Danvers, MA, USA), which detects the full-length protein (116 kDa) as well as the large caspase-cleaved fragment (89 kDa); anti-Casp3 (1 : 1000) all from CST, and anti-LC3 (2 μg/ml, Novus Biologicals, Littleton, CO, USA) anti-Gapdh (1 : 10 000, Abcam, Cambridge, UK), followed by the appropriate HRP-conjugated secondary antibodies (1 : 10000, Dako, Glostrup, Denmark). Detection was performed with ECL reagents (Pierce, Thermo Scientific, Waltham, MA, USA). The ImageJ software (open source; http://imagej.nih.gov/ij ) was used for densitometric quantification.
3
Western Blot Analysis of Protein Signaling
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After two washes with phosphate buffered saline (PBS), cells were scrapped on ice into lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA) supplemented with 1 mM PMSF and the soluble fraction was isolated after centrifugation (14000 rpm, 10 min, 4°C). Protein was quantified using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s protocol, and lysates were resolved on 8% poly-acrylamide gels and transferred onto Immobilon-P PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% milk in Tris buffered saline with 0.1% Tween-20 (TBST) for 1 hr, followed by an overnight incubation at 4°C in primary antibody (1:1000). Membranes were washed with TBST before and after exposure to goat-anti-rabbit HRP secondary antibody (1 hr; Cell Signaling) and protein were visualized using Kodak 1D Image Analysis Software 3.6 and a Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA). Primary antibodies used for western blotting include phospho-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), phospho-AKT, AKT, phospho-JNK, phospho-IKK, phospho-NF-κB, phospho-elF-2α, and phospho-TAK1 were obtained from Cell Signaling, anti-GPR120 was obtained from Abcam and anti-β-actin and anti-TNFα were obtained from Sigma.
4
Quantitative Western Blot Analysis
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Protein samples were prepared from liver homogenates in Laemmli sample buffer, run on SDS-polyacrylamide gels (4-15% TGX stain-free gel, Bio-Rad), and transferred to the polyvinylidene difluoride (PVDF) membrane. The membranes were blocked, incubated with primary antibodies overnight at 4 °C, followed by secondary antibodies, and developed using the chemiluminescence imaging system (Bio-Rad). Following primary antibodies were used: OPA1 (BD Biosciences, 612606; 1:1000); caspase-3 (Cell Signaling, 9662; 1:1000), PARP-1 (Cell Signaling, 9542; 1:1000), β-actin (Sigma, A1978; 1:40000), TOM20 (Proteintech, 11802-1-AP; 1:1000), cytochrome c (BD Biosciences, 556432; 1:5000), elF2α (Cell Signaling, 9722; 1:1000), phospho-elF2α (Cell Signaling, 9721; 1:1000), FGF21 (Proteintech, 26272-1-AP; 1:1000), LC3 A/B (Cell Signaling, 4108; 1:1000), PGC1α (Invitrogen, PA5-38022; 1:500), OMA1 (Santa Cruz, sc-515788; 1:100), and mitochondria total OXPHOS rodent WB cocktail (Abcam, ab110413; 1:1000). JNK (Cell Signaling, 9252; 1:1000), p-JNK-Thr183/Tyr185 (Cell Signaling, 9255; 1:500), MCU (Sigma, HPA016480; 1:1000), NCLX (Proteintech, 21430-1-AP; 1:1000), CypD (Proteintech, 18466-1-AP; 1:1000), MnSOD (BD Biosciences, 611580; 1:1000), GPx1/2 (Santa Cruz Biotechnology, sc-133160; 1:200), and CYP2E1 (Proteintech, 19937-1-AP; 1:300).
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