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Methods Mol Biol. 2019;1855:93-100. doi: 10.1007/978-1-4939-8793-1_9.

Isoelectric Focusing on Non-Denaturing Flatbed Gels.

Author information

1
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA. biji-kurien@omrf.org.
2
Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA. biji-kurien@omrf.org.
3
Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA. biji-kurien@omrf.org.
4
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
5
Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.
6
Department of Veterans Affairs Medical Center, Oklahoma City, OK, USA.

Abstract

Isoelectric focusing (IEF) serves as a very useful procedure for cell protein separation and characterization. We have used this method to study antibody clonotype changes. Here we discuss the use of a sensitive native flatbed isoelectric focusing method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus, who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera samples collected over several years were used for analysis using flatbed IEF. Following electrofocusing, the gel is analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane to determine oligoclonality of the anti-Ro 60 containing sera.

KEYWORDS:

Affinity immunoblotting; Clonotype distribution; Flatbed IEF; Ro 60 autoantigen; Systemic lupus erythematosus

PMID:
30426410
DOI:
10.1007/978-1-4939-8793-1_9
[Indexed for MEDLINE]

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