Biochemistry Core

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.

Email Us

Learn More

Services

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

Equipment

  • 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

Publications

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.

Meet Our Team

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


Sanford Health News

Classes & Events