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Biochemistry

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Nanyang Technological University

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

7.2K Views

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08:12 min

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January 3rd, 2019

DOI :

10.3791/58697-v

January 3rd, 2019

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Aleksandra Skrajna¹, Xiao-Cui Yang¹, Zbigniew Dominski¹^,²

¹Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, ²Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill

Summary

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Explore More Videos

Single step Purification

Macromolecular Complexes

RNA Protein Complexes

Mass Spectrometry

RNA Binding Sites

Photo cleavable Linker

Nuclear Extract

Streptavidin Agarose Beads

Adaptor PCB Oligonucleotide

Supernatant Collection

Centrifugation Process

Chapters in this video

0:04

Title

0:51

Substrate Preparation

1:28

Complex Assembly

2:14

Immobilization of the RNA/protein Complexes on Streptavidin Beads

4:00

UV-Elution

5:08

Sample Analysis by Silver Staining Followed by Mass Spectrometry

5:59

Results: Analysis of UV-eluted Complexes

7:36

Conclusion

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