Helping Researchers Use BioID & More
The Biochemistry Core provides a centralized resource of expertise, equipment and technical capabilities that cover a variety of methods in the separation and analysis of complex biological samples. These capabilities include the BioID method to screen for protein-protein interactions, multi-analyte detection and quantification of proteins and lipid analysis by GC/MS.
This core is supported by NIH P20 GM103620 as a component of the Center for Pediatric Research.
We’ve answered the most common questions about BioID in the section below titled “Frequently Asked Questions.” If you have more questions, please contact us.
BioID is a new method to screen for candidate protein-protein interactions based on proximity-dependent biotinylation BioID Resources.
The core will provide assistance with:
- Project design
- Testing of BioID-fusion proteins
- Establishment and testing of cells expressing bioid-fusion proteins
- Large scale BioID pull-downs
- Arranging for candidate identification by mass spectrometry
- Data analysis
- Bio-Rad Protean IEF Cell
- Bio-Rad Mini-Protean Dodeca Cell
- Bio-Rad Mini-Protean Tetra Cell
- Bio-Rad Protean II Xi/XL Cell
- Bio-Rad Protean II XL Multi-Cell
- GE Healthcare Typhoon Trio+ Multi-Mode Imaging System
- Agilent 7890A Gas Chromatograph System
- Agilent 5977B Mass Spectrometry System
- Luminex 200 Multiplex Protein Analysis System
- Kim DI, Jensen SC, Noble KA, KC B, Roux KH, Motamedchaboki K, Roux KJ. An improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell. 2016 Apr 15;27(8):1188-96.
- Brudvig JJ, Cain JT, Schmidt-Grimminger GG, Stumpo DJ, Roux KJ, Blackshear PJ, Weimer JM. MARCKS Is Necessary for Netrin-DCC Signaling and Corpus Callosum Formation. Mol Neurobiol. 2018 Nov;55(11):8388-8402.
- Forred BJ, Neuharth S, Kim DI, Amolins MW, Motamedchaboki K, Roux KJ, Vitiello PF. Identification of Redox and Glucose-Dependent Txnip Protein Interactions. Oxid Med Cell Longev. 2016;2016:5829063.
- Roux KJ, Kim DI, Burke B, May DG. BioID: A Screen for Protein-Protein Interactions. Curr Protoc Protein Sci. 2018 Feb 21;91:19.23.1-19.23.15.
- Kim DI, Birendra KC, Zhu W, Motamedchaboki K, Doye V, Roux KJ. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):E2453-61.
- Birendra Kc, May DG, Benson BV, Kim DI, Shivega WG, Ali MH, Faustino RS, Campos AR, Roux KJ. VRK2A is an A-type lamin-dependent nuclear envelope kinase that phosphorylates BAF. Mol Biol Cell. 2017 Aug 15;28(17):2241-2250.
- Hussain S, Bedekovics T, Liu Q, Hu W, Jeon H, Johnson SH, Vasmatzis G, May DG, Roux KJ, Galardy PJ. UCH-L1 bypasses mTOR to promote protein biosynthesis and is required for MYC-driven lymphomagenesis in mice. Blood. 2018 Dec 13;132(24):2564-2574.
- Cain JT, Kim DI, Quast M, Shivega WG, Patrick RJ, Moser C, Reuter S, Perez M, Myers A, Weimer JM, Roux KJ, Landsverk M. Nonsense pathogenic variants in exon 1 of PHOX2B lead to translational reinitiation in congenital central hypoventilation syndrome. Am J Med Genet A. 2017 May;173(5):1200-1207.
- Kim DI, Jensen SC, Roux KJ. Identifying Protein-Protein Associations at the Nuclear Envelope with BioID. Methods Mol Biol. 2016;1411:133-46.
- Mehus AA, Anderson RH, Roux KJ. BioID Identification of Lamin-Associated Proteins. Methods Enzymol. 2016;569:3-22.
BioID is a method to screen for proximate and interacting proteins in living cells. This is accomplished by fusion of a promiscuous biotin ligase (we call it BirA*) to a protein of interest (a bait) for expression in live cells. Addition of excess biotin leads to efficient biotinyation of endogenous proteins adjacent to/interacting with the bait. These biotinylated proteins (called candidates) can be monitored by fluorescence microscopy or Western blot analysis and isolated for identification by mass spectrometry. The biological relevance of the candidates to the bait can be subsequently validated by a variety of conventional approaches.
Meet Our Team
Kyle Roux, PhD
Through his research, Dr. Kyle Roux seeks to understand the structure and function of the nuclear envelope in health and disease, and to further develop the BioID method. His work, along with other research groups, gave rise to the LINC-complex model, a structure that spans the nuclear envelope and links the nucleoskeleton to the cytoskeleton. He also investigated the post-translational processing of lamin-A, a major constituent of the nuclear lamina, both in a mouse model for Hutchinson-Gilford Progeria Syndrome, and in relation to protease and farnesyltransferase inhibitors.
Peter Vitiello, PhD
Dr. Peter Vitiello is a molecular toxicologist with a research program studying redox signaling during mammalian development, health and oxidative disease. He oversees services related to separation and identification of complex mixtures, including multi-analyte detection of proteins and GC/MS quantification of lipids.
Danielle May, MS
Danielle May received her B.S. in physiology and neuroscience from the University of California-San Diego and continued her work there to earn an M.S. in biology. She then worked at the Scripps Research Institute in La Jolla, California, learning mass spectrometry and proteomics in the Yates Lab before joining the Biochemistry Core.
Rachel Chrisopulos received her B.S. in biology with a specialization in molecular biology and physiology from the University of South Dakota. She then worked as a pesticide residue chemist at SGS in Brookings, South Dakota, for five years, working extensively with LC-MS/MS and GC-MS instrumentation before joining the Biochemistry Core.
Frequently Asked Questions
How do I get the biotin to go into solution in the cell media?
We make a 20X stock solution (1mM) of biotin (Sigma cat# B4639) in our standard tissue culture media (without serum) and use it at 1X (50uM, with serum). This 20X solution is filter sterilized and is stable at 4 deg C for at least a couple of months.
Do I have to regulate inducibly regulate expression of my BioID fusion protein?
As a rule, no. We have generated many stable cell lines that constitutively express a variety of BioID fusion proteins and utilized these for large-scale BioID pull-downs. The most effective method to regulate biotinylation by BioID is the addition of excess biotin to cell culture media. However, in some circumstances it may be ideal and/or necessary to inducibly express a BioID-fusion protein, for example if the protein is toxic.
Do I need to express a lot of BioID-fusion protein to identify interaction candidates?
In our experience, no. Although more fusion protein can equal more biotinylation of endogenous proteins, we are able to obtain a large number of uniquely biotinylated proteins (defined as those not detected in parallel pull-downs from control cells) from cells stably expressing BioID-fusion proteins in which we can barely detect the protein by immunofluorescence.
Will BioID work in my cell type/model organism?
We have applied BioID only to mammalian cells, specifically HeLa, HEK293, U2OS, C2C12 cell lines. Other groups are at various stages of applying BioID in a variety of cell types/model organisms. These include S. cerevisiae, C. elegans, Trypanosomes and Toxoplasma. Our suggestion is to try it out (and let us know if it works).
What is the effective range of biotinylation by BioID?
Please see our manuscript on this topic: Kim DI, KC B, Zhu W, Motamedchaboki K, Doye V, Roux KJ. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci USA. 2014 Jun 17;111(24):E2453-61. Pubmed PMID: 24927568
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