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Serine/threonine protein kinase which is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy and stress signals. MTOR directly or indirectly regulates the phosphorylation of at least 800 proteins. Functions as part of 2 structurally and functionally distinct signaling complexes mTORC1 and mTORC2 (mTOR complex 1 and 2). Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. This includes phosphorylation of EIF4EBP1 and release of its inhibition toward the elongation initiation factor 4E (eiF4E). Moreover, phosphorylates and activates RPS6KB1 and RPS6KB2 that promote protein synthesis by modulating the activity of their downstream targets including ribosomal protein S6, eukaryotic translation initiation factor EIF4B, and the inhibitor of translation initiation PDCD4. Stimulates the pyrimidine biosynthesis pathway, both by acute regulation through RPS6KB1-mediated phosphorylation of the biosynthetic enzyme CAD, and delayed regulation, through transcriptional enhancement of the pentose phosphate pathway which produces 5-phosphoribosyl-1-pyrophosphate (PRPP), an allosteric activator of CAD at a later step in synthesis, this function is dependent on the mTORC1 complex. Regulates ribosome synthesis by activating RNA polymerase III-dependent transcription through phosphorylation and inhibition of MAF1 an RNA polymerase III-repressor.
1. HOW DID OUR ANTIBODY PERFORM? CUSTOMER REVIEW: mTOR Polyclonal Antibody (STJ94280)
2. WHAT WAS USED? Primary antibody: STJ94280. mTOR Polyclonal Antibody. 100ul Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot. Image: In HeLa and Jurkat cell line, there were target bands detected by STJ94280 mTOR Polyclonal Antibody.
3. WHAT WAS THE PROTOCOL? Treatment of materials: When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors (Sigma) and equal volume of 2X loading buffer were added for 10 min water bath at 100?C. 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 60 min. Blocking: 1X TBST with 5% skimmed milk powder for 2 hr. Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4?C for overnight. Membrane wash: 1X TBST wash for 3 times. Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1:10000 dilution ratio, for 1.5 hr. Membrane wash: 1X TBST wash for 3 times. Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).
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 60 min. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 HeLa lysate 10 ug STJ94280. mTOR Polyclonal Antibody 1:1000 1:10000 288KD 35S 2 Jurkat lysate 10 ug 1:10000 288KD 35S
5. WHAT DID THE CUSTOMER THINK? Testing results were provided by BPI laboratory. They stated that ¡°In HeLa and Jurkat cell line, there were target bands detected by STJ94280 mTOR Polyclonal Antibody¡±.
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