Improving sensitivity of the electrophoretic mobility shift assay by restricting tissue phosphatase activities.
Elucidating DNA-protein interactions at the molecular level is a prerequisite in understanding the way a transcriptionally active gene is regulated in various tissues. A number of techniques are presently available for this particular type of analyses, of which, the electrophoretic mobility shift assay (EMSA) is certainly the preferred one and likely the most sensitive and powerful. EMSA is widely used in combination with nuclear extracts obtained from cultured cells. However, its use with crude extracts prepared from whole animal tissues is still restricted. The restriction is partly due to numerous enzymatic activities, such as proteases and deacetylases, that strongly interfere with the EMSA's sensitivity. In addition to endogenous proteases, which can be inhibited by the addition of protease inhibitors to the buffers used, crude nuclear extracts obtained from whole animal tissues are often contaminated with considerable amounts of highly active endogenous phosphatases. Some of these enzymes substantially interfere with the ability of the EMSA to unravel specific DNA-protein interactions by removing the 5'-[32P] end-labeled phosphate of the DNA probe used for the binding assay. In this study, we evaluated whether we can restore the sensitivity of the EMSA by modifying the conditions under which the DNA-protein binding reaction is normally performed. Dephosphorylation by endogenous tissue phosphatases of the labeled probe used in our assays was drastically prevented by simply reducing both the temperature at which the binding reaction was normally performed and the time allowed for the DNA-protein interaction to occur.
Laniel
M A
MA
CHUL Research Center, Ste-Foy, Canada.
Guérin
S L
SL
D000818:Animals; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004789:Enzyme Activation; D008297:Male; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D015342:DNA Probes; D004268:DNA-Binding Proteins; D004247:DNA; D010744:Phosphoric Monoester Hydrolases