Isoelectric point mobility shift assay for rapid screening of charged and uncharged ligands bound to proteins

Petra Kempná, Rita Cipollone, Luis Villacorta, Roberta Ricciarelli, Jean Marc Zingg

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Three human proteins (hTAP1, hTAP2 and hTAP3) that are related to the yeast phosphatidylinositol/phosphatidylcholine transfer protein SEC14p were recently cloned in our laboratory. These proteins contain a relatively large hydrophobic pocket, the so called CRAL-TRIO domain, which is present also in other human proteins, such as CRALBP, α-TTP and MEG2. The CRAL-TRIO domains in these proteins bind ligands such as retinaldehyde, tocopherols and polyphosphoinositides, respectively. To screen for potential hTAPs ligands, we developed a semi-quantitative isoelectric point mobility shift assay (IPMS-assay) that allows assessing the binding of potential hydrophobic ligands to proteins. Purified proteins occupied with a charged ligand migrate differently on isoelectric focusing gels when compared with free protein. Competition of bound charged ligands with uncharged ones reverses the mobility shift, so that the relative affinities of the two ligands to the protein can be estimated.

Original languageEnglish (US)
Pages (from-to)103-107
Number of pages5
JournalIUBMB life
Volume55
Issue number2
DOIs
StatePublished - Feb 1 2003
Externally publishedYes

Keywords

  • Binding assay
  • Human tocopherol associated proteins (hTAP)
  • Isoelectric focusing
  • Lipid-protein interaction
  • Phospholipids
  • SEC14
  • Tocopherol

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics
  • Clinical Biochemistry
  • Cell Biology

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