Isoelectric Focusing on Non-Denaturing Flatbed Gels

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

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.

MeSH terms

  • Collodion / chemistry
  • Humans
  • Immunoblotting
  • Isoelectric Focusing
  • Lupus Erythematosus, Systemic / blood*
  • Lupus Erythematosus, Systemic / metabolism
  • Ribonucleoproteins / blood
  • Ribonucleoproteins / isolation & purification*

Substances

  • Ribonucleoproteins
  • SS-A antigen
  • Collodion