Chapter title |
On-Membrane Renaturation of Recombinant Ro60 Autoantigen by Calcium Ions
|
---|---|
Chapter number | 26 |
Book title |
Detection of Blotted Proteins
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2718-0_26 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2717-3, 978-1-4939-2718-0
|
Authors |
Biji T. Kurien, Michael P. Bachmann, Kurien, Biji T., Bachmann, Michael P. |
Abstract |
Calcium plays an important role in many biochemical processes in different cell types. This divalent cation interacts with specific calcium-binding proteins that serve as calcium sensors and regulatory proteins to mediate its function. Previously, we found that calcium was involved in the protein-protein interaction observed between Ro60 multiple antigenic peptides and Ro60 autoantigen. Since calcium bound Ro60 multiple antigenic peptides, we hypothesized that it would renature human recombinant Ro60 on a protein blot. We found that anti-Ro60 antibodies bound significantly higher to the recombinant Ro60 antigen that was incubated with calcium compared to that incubated without calcium on a polyvinylidene fluoride (PVDF) blot. Since the immunological epitopes of Ro60 are mainly conformational, we believe that calcium induced a more native tertiary structure in recombinant Ro60 autoantigen following blotting to a PVDF membrane. |
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