The BioID assay was performed essentially as described (25 (link)). Briefly, N-terminal BirA-FOXB2 and BirA plasmid were transfected into 293T cells using Lipofectamine 2000 (Thermo Fisher). After transfection, cells were treated with 50 µM biotin. Biotinylated proteins were precipitated with streptavidin beads (GE Healthcare) and digested using spectrometry-grade trypsin (Thermo Fisher). BioID samples were analyzed by mass spectrometry using an Easy nano LC II HPLC interfaced with a nanoEasy spray ion source (Thermo Fisher) connected to an Orbitrap Velos Pro mass spectrometer (Thermo Fisher).
Protocol detail
BioID Assay for Protein Identification
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
293t cells
Assay
Biotin
Cells
Hplc
Lipofectamine 2000
Mass spectrometry
Plasmid
Proteins
Spectrometry
Streptavidin
Transfection
Trypsin
Corresponding Organization :
Other organizations : Linköping University
Top 5 similar protocols
Variable analysis
independent variables
- Transfection of N-terminal BirA-FOXB2 and BirA plasmid into 293T cells
- Treatment of cells with 50 µM biotin
- Biotinylated proteins precipitated with streptavidin beads
- Digestion of biotinylated proteins using trypsin
- Analysis of BioID samples by mass spectrometry
- Use of Lipofectamine 2000 for transfection
- Use of spectrometry-grade trypsin for digestion
- Use of Easy nano LC II HPLC and nanoEasy spray ion source connected to an Orbitrap Velos Pro mass spectrometer for mass spectrometry analysis
- Positive control: BirA plasmid
- Negative control: Not explicitly mentioned
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!