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eIF2 is an initiation factor found within eukaryotes. It mediates binding of tRNA to the ribosome in a GTP dependent manner. The alpha subunit is the main target for phosphorylation at Serine-51, and is considered the regulatory subunit of this trimeric (formed of eIF2a, eIF2b and eIF2y).
1. HOW DID OUR ANTIBODY PERFORM? CUSTOMER REVIEW: Phospho-eIF2alpha (S51) Polyclonal Antibody (STJ90253)
2. WHAT WAS USED? Primary Antibody: Provider: Dilution ratio: Secondary Antibody: Application: Materials for validation: STJ90253 Phospho-eIF2alpha (S51) Polyclonal Antibody St John’s Laboratory 1:500 Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate (Sigma) Western Blot. Murine intestinal longitudinal muscle myenteric plexus (LMMP)
3. WHAT WAS THE PROTOCOL? Treatment of materials: LMMP were homogenized using a polytron tissue grinder in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.25% wt/vol sodium deoxycholate, 0.1% Nonidet P- 40, 100 M NaVO4) with protease inhibitors (Sigma). Samples were treated with loading buffer for 5 min in water bath at 100¡ãC. Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min. Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V, 90 min. Blocking: 1X TBST with 5% bovine serum albumin for 30 min. Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% bovine serum albumin, 4?C for overnight. Membrane wash: 1X TBST washes for 3 times. Secondary antibody probing: Secondary antibody was added to the solution of 1X TBST with 5% bovine serum albumin with 1:2000 dilution ratio, for 45 min. Membrane wash: 1X TBST washes for 3 times. Visualization: ECL visualization for 5 min. Images were captured using Hyper Film MP (GE Healthcare).
4. WHAT WAS THE PROTOCOL? Gel electrophoresis information£º10% polyacrylamide gel, constant voltage 160 V, 60 min. Transfer information£º0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 90 min. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 LMMP from C57Bl6J young mouse 20 ?g STJ90253 Phospho- eIF2? (S51) 1:500 1:2000 ~30KD 10S 2 LMMP from C57Bl6J aged mouse
5. WHAT DID THE CUSTOMER THINK? Testing results were provided by Ignazio Castagliuolo from University of Padova, Italy. He stated that ¡°In LMMP isolated from both young and aged C57Bl6J mice, there were target bands detected by STJ90253 Phospho-eIF2 (S51)
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