A Robust Method to Store Complement C3 With Superior Ability to Maintain the Native Structure and Function of the
Anna Adler1, Vivek Anand Manivel1, Karin Fromell1
1Rudbeck Laboratory, Department of Immunology, Genetics and Pathology (IGP), Uppsala University, Uppsala, Sweden.
Frontiers in Immunology
|May 20, 2022
Summary
Storing native complement component 3 (C3) by precipitation and freezing prevents its degradation into C3(H2O). This new method preserves C3 function, overcoming a major challenge in complement research.
Area of Science:
- Immunology
- Protein Biochemistry
Background:
- Complement component 3 (C3) is crucial for immune responses but is highly labile.
- Spontaneous hydrolysis of its internal thioester leads to the formation of inactive C3(H2O).
- Existing storage methods for C3 are unreliable, potentially compromising research findings.
Purpose of the Study:
- To define optimal storage conditions for native C3.
- To develop a robust method minimizing C3(H2O) generation.
- To establish conditions for consistent C3(H2O) formation for experimental use.
Main Methods:
- Precipitation of native C3 at its isoelectric point in low ionic strength buffer.
- Freezing the precipitated C3 at -80°C for storage.
- Analysis of C3(H2O) formation using cation exchange chromatography.
- Assessment of hemolytic activity via classical pathway hemolytic assay.
Main Results:
- Precipitation and freezing of native C3 effectively prevented C3(H2O) generation and preserved protein function.
- Incubation of C3 in a buffer at pH 11.0 consistently induced C3(H2O) formation.
- The developed method offers a reliable way to store C3 without functional loss.
Conclusions:
- Optimal storage conditions for native C3 involve precipitation at the isoelectric point followed by freezing at -80°C.
- This method significantly reduces C3(H2O) formation, ensuring protein stability and function.
- Defined conditions allow for both stable C3 storage and controlled generation of C3(H2O).
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