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This page is meant to provide information to those seeking to learn more about BioID.

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.



Published Resources

Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux KJ. An improved smaller biotin ligase for BioID proximity labeling.. 2016.

Kim DI, Jensen SC, Roux KJ. Identifying Protein-Protein Associations at the Nuclear Envelope with BioID. Methods in Molecular Biology. 2016.

Mehus A, Anderson RH, Roux KJ. BioID Identification of Lamin-Associated Proteins. Methods in Enzymology, 2016.

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 

Roux KJ, Kim DI, Raida M, Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 2012 Mar 19;196(6):801-10. PubMed PMID: 22412018

A commentary on this manuscript, written by Ben Short, can be found at 

This manuscript has been recommended/reviewed by Faculty 1000 at

Methodological Resources

BioID plasmids: We have deposited at Addgene, the non-profit plasmid repository, two mammalian expression plasmids for BioID.

These plasmids are designed to facilitate N-terminal or C-terminal BioID fusions. Maps and sequence are available at the Addgene website.

Mass Spectrometry: For analysis of large-scale BioID pull-downs we have utilized the Sanford-Burnham Proteomics Facility  

Frequently Asked Questions

These represent the most common questions we have received about BioID. Please contact us with your questions and we may include them here.

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