Constructs, antibodies, and recombinant proteins derived from earlier studies2 ,18 are fully described in Supplementary Methods. Antibodies to Bat3 and Ubl4A were produced in rabbits immunized with recombinant Bat3 fragment (residues 1-250) or full-length Ubl4A, respectively. TRC35 antibodies were against a synthetic peptide (NH2-CGSPIELD-COOH) conjugated to KLH. In vitro transcription, translation in reticulocyte lysate, sucrose gradient fractionation, chemical crosslinking, and immunoprecipitation was as before2 . RNCs were generated by transcription and translation of PCR products lacking a stop codon and sucrose gradient purified for use in the capture assay. CTAB precipitation of tRNA-associated polypeptides has been described28 .
Protocol detail
Recombinant Protein Purification Protocols
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Antibodies
Assay
Ctab
Fractionation
Immunoprecipitation
Peptide
Polypeptides
Rabbits
Recombinant proteins
Reticulocyte
Stop codon
Sucrose
Transcription
Trna
Corresponding Organization :
Other organizations : National Institutes of Health, Peking University, Eunice Kennedy Shriver National Institute of Child Health and Human Development, University of Chicago
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Variable analysis
independent variables
- Recombinant Bat3 fragment (residues 1-250)
- Full-length Ubl4A
- Synthetic peptide (NH2-CGSPIELD-COOH) conjugated to KLH
- Antibodies to Bat3 and Ubl4A
- TRC35 antibodies
- In vitro transcription
- Translation in reticulocyte lysate
- Sucrose gradient fractionation
- Chemical crosslinking
- Immunoprecipitation
- CTAB precipitation of tRNA-associated polypeptides
- Positive control: Not specified
- Negative control: Not specified
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