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Isoelectric Point Mobility Shift Assay for Rapid Screening of Charged and Uncharged Ligands Bound to Proteins
Author(s) -
Kempná Petra,
Cipollone Rita,
Villacorta Luis,
Ricciarelli Roberta,
Zingg JeanMarc
Publication year - 2003
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/1521654031000095756
Subject(s) - isoelectric point , chemistry , affinities , isoelectric focusing , ligand (biochemistry) , biochemistry , plasma protein binding , dna binding protein , receptor , gene , enzyme , transcription factor
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. IUBMB Life, 55: 103‐107, 2003