Chapter title |
Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane
|
---|---|
Chapter number | 17 |
Book title |
Detection of Blotted Proteins
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2718-0_17 |
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 |
Antibodies directed against ribonucleoprotein (RNP) particles are observed in systemic lupus erythematosus. Ro RNP particle is one such target. It is composed of a 60 kDa protein (Ro 60 or SS-A) that is non-covalently associated with at least one of the four short uridine-rich RNAs (the hY RNAs). Previously, we showed that multiple antigenic peptides (MAPs) made from the sequence of the Ro 60 autoantigen could be used, using double-immunodiffusion studies, enzyme-linked immunosorbant assay, affinity chromatography, and surface plasmon resonance, to show intramolecular and intermolecular protein-protein interaction within the Ro 60 RNP particle. We also observed that calcium is important in mediating this interaction. We hypothesized, therefore, that 60 kDa Ro is a calcium-binding protein. To investigate this, we electrophoresed 60 kDa Ro MAPs, transferred them to PVDF membrane, and assayed calcium binding using the Quin-2 system. Several Ro 60 MAPs were found to bind calcium using this assay, as well as bovine serum albumin, another calcium-binding protein. However, a MAP constructed from the Sm autoantigen did not bind to calcium. These data, along with our observation regarding the involvement of calcium in protein-protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, makes us propose that Ro 60 antigen is a calcium-binding protein. |
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