77 Micro-IP Rapid Isolation of Protein Complexes from Cellular Lysates

Faculty Mentor: Peter Shen (Biochemistry, University of Utah)

One of the most important requirements in many protein biochemistry experiments is the ability to isolate and purify proteins of interest [1,4]. Traditional methods typically aim to generate large quantities of proteins; however, such methods typically rely on long, multi-day experiments and are only effective for well-behaved proteins. Co-immunoprecipitation (co-IP) is one common tool to purify protein samples and can give insight into protein-protein interactions. Co-IPs performed from cell lysates can also recover native post-translational modifications and binding partners compared to traditional methods [2-4]. However, current co-IP methods are time-consuming, require extensive amounts of starting material, and may contain significant amounts of non-specific contamination. I have refined a novel approach termed “micro-IP” that enables micro-scale purifications of native protein complexes from cell lysates that require less than one gram of starting material. Micro-IP utilizes neodymium magnets to immobilize magnetic resin beads coated with specific antibodies. The beads are flowed through thin tubing and become clustered by the neodymium trap. Cell lysate is then flowed through the tubing and over the resin, which captures target proteins through interactions with the antibodies coated on the resin. This results in a 4-fold decrease in time, and a 5-folddecrease in the amount of sample needed, and yields are purer than standard co-IP methods. These improvements can therefore pave the way for faster, cheaper, and cleaner protein purification. This method may be used to discover new biochemical interactions, mechanisms, and pathways.

References

[1] Xia, Hui, et al. ‘Microfluidic Based Immunosensor for Detection and Purification of Carbonylated Proteins’.Biomedical Microdevices, 15, no. 3, June 2013, pp. 519–30. DOI.org (Crossref), https://doi.org/10.1007/s10544-013-9751-2.

[2] Cooney, Ian, et ‘Lysate-to-Grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging’. BIO-PROTOCOL, vol. 13, no. 2, 2023. DOI.org (Crossref), https://doi.org/10.21769/BioProtoc.4596.

[3] Markham, Kelly, et al. ‘Co-Immunoprecipitations Revisited: An Update on Experimental Concepts and TheirImplementation for Sensitive Interactome Investigations of Endogenous Proteins’. Analytical and Bioanalytical Chemistry, vol. 389, no. 2, Sept. 2007, pp. 461–73. DOI.org (Crossref), https://doi.org/10.1007/s00216-007-1385-x.

[4] Pazour, Gregory ‘Immunoprecipitation to Examine Protein Complexes’. Methods in Cell Biology, vol. 91, Elsevier, 2009, pp. 135–42. DOI.org (Crossref), https://doi.org/10.1016/S0091- 679X(08)91008-6.