Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7 (link),8 (link). Recombinant active S6K132 (link) and RSK1–3 were from Cell Signaling; Akt1, and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 (link) to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N-terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33 (link). Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen).
Recombinant active s6k132
Manufactured by Cell Signaling Technology
Recombinant active S6K132 is a purified, recombinant protein that represents the catalytically active form of the 70 kDa ribosomal protein S6 kinase (S6K1). S6K1 is a serine/threonine protein kinase that phosphorylates the S6 protein of the 40S ribosomal subunit, which is involved in the regulation of protein synthesis.
Automatically generated - may contain errors
2 protocols using recombinant active s6k132
1
Purification and Mutagenesis of EPRS Linker
2
Purification and Mutagenesis of EPRS Linker
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7 (link),8 (link). Recombinant active S6K132 (link) and RSK1–3 were from Cell Signaling; Akt1, and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 (link) to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N-terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33 (link). Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!