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Protocols

Immunopurification protocol for the pulldown of endogenous RNA-binding proteins ("RIP") and co-purification of (associated) RNA molecules from human cell extracts. RNA prepared according to this protocol can be analyzed using microarrays or (possibly) sequencing techniques, allowing the identification of the RNA targets of the RBP under study. This protocol has been optimized for the immunopurification of endogenous human PUM1 and PUM2 proteins from HeLa S3 cells.
Protocol by Alessia Galgano.
Reference:

  1. Galgano A, Forrer M, Jaskiewicz L, Kanitz A, Zavolan M, and Gerber AP. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system. PLoS One. 2008 Sep 8;3(9):e3164. DOI:10.1371/journal.pone.0003164 | PubMed ID:18776931 | HubMed [Paper17]


Immunopurification protocol for the pulldown of tagged ribosomal proteins and the co-purification of (associated) RNA molecules from yeast cell extracts. RNA prepared according to this protocol can be analyzed using microarrays or (possibly) sequencing techniques. This protocol has been optimized for the study of yeast ribosomes through the immunopurification of the large ribsomal subunit protein Rpl16a.
Protocol by Regula Halbeisen.
References:

  1. Halbeisen RE and Gerber AP. Stress-dependent coordination of transcriptome and translatome in yeast. PLoS Biol 2009 May 5; 7(5) e105. doi:10.1371/journal.pbio.1000105. [Original_Research_Article]
  2. Halbeisen RE, Scherrer T, and Gerber AP. Affinity purification of ribosomes to access the translatome. Methods. 2009 Jul;48(3):306-10. DOI:10.1016/j.ymeth.2009.04.003 | PubMed ID:19398006 | HubMed [Method]