Estrogen receptor preparation effects on the receptor–DNA interaction by surface plasmon resonance

Abstract

Up to now, several studies have investigated estrogen receptor (ER)-estrogen response element (ERE) interaction using biosensors such as surface plasmon resonance. These strategies have aimed to understand the molecular mechanism of such interaction as well as the effect of the ligand on this interaction. These approaches start to be used to determine the mechanisms of protein/DNA interaction, in particular in the context of drug discovery or environmental applications. However, some physical and biochemical parameters (incubation time, temperature, protease inhibitor cocktail, and bovine serum albumin (BSA)) are not completely described in the literature and could deeply modify the obtained results. This paper aims to focus not only on the preliminary steps of sample preparation such as protein thawing and incubation conditions (time and temperature) but also on the evaluation of protease inhibitor cocktail and BSA effect on the measurement of ER-ERE interactions.

Critical parameters in surface plasmon resonance biosensor development: The interaction between estrogen receptor and estrogen response element as model

“…As found in previous studies [1,3,8], the temperature and time of sample conservation induce a modification of the ER binding level. Indeed, the ER binding level decreased by between 114 and 59…”

“…These same parameters can also affect ER/ERE interaction, as previously shown [1]. Other factors, such as the protein thawing method or the ER/E2 mixing temperature are also essential [3].…”

“…RU ( Figure 7A-B For the first set of injections ( Figure 7A) the E2 dose effect on the ER binding is observed to produce nearly a two-fold increase in the SPR signal from 128 RU to 225 RU ( Figure 7B). The difference between the loss of binding of ER (59 RU) and the last point of the dose curve is due to the presence of E2, which permits greater stability of ER in ambient temperature [1,3]. During the second set of injections ( Figure 7C-D), the overall ER binding level decreases even in the presence of E2.…”

“…Most relied on a comparison between ER on its own, and ER with the studied molecule [2, 8, 10, 14-17, 19, 19]. The binding level is the most commonly used parameter for comparisons, however another indicator that can be used is the fold change in binding level [2,3,10]. Some publications have also calculated affinity constants between ER and ERE depending upon the treatment.…”

“…The difference in the KD varies by nearly two orders of magnitude between manipulations. These variations may be due to the difficulties occurring with A c c e p t e d m a n u s c r i p t the conservation of ER depending upon the ER preparation and experiment conditions [1,3,20]. This makes it difficult to calculate reliable affinity constants for ER/ERE interaction.…”