Published: Vol 7, Iss 15, Aug 5, 2017 DOI: 10.21769/BioProtoc.2430 Views: 10908
Reviewed by: Jyotiska ChaudhuriLiang LiuAnonymous reviewer(s)
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
Related protocols
Proteome Birthdating: A Single-Sample Approach for Measuring Global Turnover Dynamics and “Protein Age”
Michael E. Meadow [...] Sina Ghaemmaghami
May 5, 2025 807 Views
ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis
Qiqing He [...] Fuchu He
Jul 20, 2025 1451 Views
Fluorescence Polarization-Based High-Throughput Screening Assay for Inhibitors Targeting Cathepsin L
Keyu Guo [...] Shuyi Si
Jul 20, 2025 817 Views
Abstract
This protocol describes a method for purifying glycosylated FLAG-tagged secreted proteins with disulfide bonds from mammalian cells. The purified products can be used for various applications, such as ligand binding assays.
Keywords: Secreted proteinBackground
E. coli is one of the organisms of choice for production of recombinant proteins from both prokaryotes and eukaryotes. The major advantages of bacterial expression systems are high productivity and low cost. However, mature proteins are essential for functional analyses (e.g., ligand binding assays). Most secreted eukaryotic proteins undergo post-translational modification by covalent glycan linkage and formation of disulfide bonds in the endoplasmic reticulum. These covalent modifications are essential for the general stability and folding of the secreted mature proteins. Therefore, the best method for production of mammalian secreted proteins is the use of a mammalian host to ensure the production of recombinant proteins that have undergone proper post-translational modifications. In the following protocol, we have described the detailed procedure for purification of secreted proteins from mammalian cells. This protocol is for production of FLAG-tagged proteins, but should be applicable to that of other tagged (e.g., His-tag) proteins as well.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was adapted from Chen et al. (2017). This work was supported by JSPS KAKENHI (Young Scientists A) Grant Number 16H06167, MEXT KAKENHI (Scientific Research on Innovative Areas) Grant Number 16H01194 (to E. I.); a European Molecular Biology Organization Fellowship (to C.C.); the Medical Research Council, UK; and the European Research Council (Advanced Grant 269058) grant to M.d.B.
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Itakura, E., Chen, C. and de Bono, M. (2017). Purification of FLAG-tagged Secreted Proteins from Mammalian Cells. Bio-protocol 7(15): e2430. DOI: 10.21769/BioProtoc.2430.
Category
Biochemistry > Protein > Isolation and purification
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link