Abstract
RNA binding proteins (RBPs) play a crucial role in regulating gene expression at the post-transcriptional level at multiple steps including pre-mRNA splicing, polyadenylation, mRNA stability, mRNA localization and translation. RBPs regulate these processes primarily by binding to specific sequence elements in nascent or mature transcripts. There are several hundreds of RBPs in plants, but the targets of most of them are unknown. A variety of experimental methods have been developed to identify targets of an RBP. These include RNA immunoprecipitation (RIP), UV cross-linking and immunoprecipitation (CLIP) and many variations of CLIP (e.g. PAR-CLIP, iCLIP). These approaches depend on immunoprecipitation of RNAs bound to a specific RBP using an antibody to that RBP. Electrophoretic mobility shift assay (EMSA), also called gel shift assay, has been used to analyze protein-nucleic acid interactions. It is a simple and powerful method to analyze protein-RNA/DNA interactions. In RNA EMSA, RNA-protein complexes are visualized by comparing the migration of RNA in the presence of a protein. Generally, in RNA EMSA a specific RNA sequence is used to analyze its interaction with a protein. In vitro transcribed 32P labeled or chemically synthesized RNA with a fluorescent tag is incubated with or without the protein of interest and the reaction mixture is then run on native polyacrylamide gel electrophoresis. RNA-Protein complexes migrate slowly as compared to free RNA, which can be visualized using an imaging system. In addition to test binding of an RBP to RNA, EMSA is also used to map the region in RNA and/or protein that is involved in interaction. Furthermore, the binding affinity can also be quantified using EMSA.
Materials and Reagents
Note: All work should be done in an RNase free environment, using only sterile, RNase free solutions and materials.
Equipment
Procedure
Note: We routinely produce RNA probes by in vitro transcription using a linearized pGEM clone that contains the DNA corresponding to RNA of interest. pGEM vector has T7 and SP6 promoters. Depending on the orientation of insert DNA either T7 polymerase or SP6 polymerase is used in in vitro transcription to generate RNA probe.
Notes
Recipes
Note: All buffers should be prepared with RNase free water.
Acknowledgments
This protocol was adapted from Thomas et al. (2012). This work was supported by a grant from the US National Science Foundation.
References
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Hi,we normally use 100ug/ml final concentration of BSA for this, but based on your conditions you have to optimize this. And also your protein should be purified as much as possibleNot exceed more than 15ul of loading volume, if you increase the loading volume, you are increasing the salt concentration and this will effect on PH and finally it will create problem on running condition. If possible add 1 ul tRNA in addition Heparin sulfate. good luck
Hi Juseok,Sorry to take so long to get back to you. Your above question seems to be a kind of general question. We would suggest that more specific question/comment related to this protocol could be addressed more effectively by our authors or other experts.Thanks,Bio-protocol Editorial Board
Hi, Please see Prof. Palusa's response to your question above.Thanks,Bio-protocol Editorial Borad