Fluorescent microplate-based analysis of protein-DNA interactions. II: Immobilized DNA.
A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.
Zhang
Zhan-Ren
ZR
Aston University, Birmingham, UK.
Hughes
Marcus D
MD
Morgan
Leonie J
LJ
Santos
Albert F
AF
Hine
Anna V
AV
D000327:Adsorption; D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry*; D019544:Equipment Failure Analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D004247:DNA