Fluorescent microplate-based analysis of protein-DNA interactions. I: Immobilized protein.
A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.
Zhang
Zhan-Ren
ZR
Aston University, Birmingham, UK.
Palfrey
David
D
Nagel
David A
DA
Lambert
Peter A
PA
Jessop
Robert A
RA
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