Isoelectric point mobility shift assay for rapid screening of charged and uncharged ligands bound to proteins.
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, alpha-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.
Kempná
Petra
P
Institute of Biochemistry and Molecular Biology, University of Bern, Bühlstrasse 28, 3012 Bern, Switzerland.
Cipollone
Rita
R
Villacorta
Luis
L
Ricciarelli
Roberta
R
Zingg
Jean-Marc
JM
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008024:Ligands; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D008024:Ligands; D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; C073119:TAP1 protein, human; C405036:thyrotropin-releasing hormone-associated peptide 3, human