Photoaffinity labeling on magnetic microspheres (PALMm) methodology for topographic mapping: preparation of PALMm reagents and demonstration of biochemical relevance.
Photoaffinity labeling (PAL) is a technique widely used for identifying the binding-site within proteins. Although the classic method is both versatile and powerful, it suffers significant disadvantages, such as the need to radiolabel the PAL ligand, and the need to conduct highly complicated separations of both the labeled protein and the labeled peptides derived from it. Here, we propose a novel and universal methodology--Photo-Affinity Labeling on Magnetic microspheres (PALMm) designed to simplify and shorten the PAL protocol. In this context, we describe the preparation of PALMm reagents and the evaluation of their biochemical relevance regarding two ATP-binding enzymes: hexokinase and apyrase.
Halbfinger
Efrat
E
Department of Chemistry, Gonda-Goldschmied Medical Research Center, Bar-Ilan University, Ramat-Gan 52900, Israel.
Gorochesky
Karine
K
Lévesque
Sébastien A
SA
Beaudoin
Adrien R
AR
Sheihet
Larisa
L
Margel
Shlomo
S
Fischer
Bilha
B
D001081:Apyrase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D015195:Drug Design; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008280:Magnetics; D008863:Microspheres; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels; D013056:Spectrophotometry, Ultraviolet
D009711:Nucleotides; D019920:Photoaffinity Labels; D006593:Hexokinase; D001081:Apyrase