Abstract
Because of the rapidly expanding need for higher sample throughput in drug discovery, automation of corresponding biochemical analyses is desirable. In particular, automation of protein quantitation is crucial since its results are used extensively. Recently, a single-reagent fluorescent protein quantitation method (NanoOrange®) with attractive performance attributes has become available. While it can potentially be automated with liquid handling workstations, several of this method's reaction parameters need to be optimized.
We studied the time and a temperature dependence of the NanoOrange protein quantitation reaction in ninety-six well black microplates using either a temperature-regulated hot block or a microwave oven as heat sources. Fluorescence of the NanoOrange reaction was quantified with a multimode microplate spectral scanner.
Time-dependent heating profiles of filled microplates placed on hot blocks at fixed temperatures (45, 55, 65, 75, and 95°C) revealed temperature differences of 4–7°C cooler for the outside wells compared to the inner wells, however the maximum well temperature did not exceed 65°C. Similar time-temperature studies of microwave-heated microplates revealed an equilibrium temperature of 45–49°C that was 10–16°C lower than microplates that were block heated.
The bovine serum albumin (BSA): NanoOrange standard curves created using a hot block increased in slope from 45°C to 55°C, but then remained constant from 65 to 85°C. Fluorescence of the BSA: NanoOrange standard curve created using a microwave oven was about half the magnitude of the hot block-derived curves, possible reflecting a lower energy transfer rate of the microwave oven. We conclude that the NanoOrange protein quantitation method can be automated if a microplate-compatible hot block with a 65–85°C surface can heat the microplate for minimum of 15 min prior to quantifying the reaction's fluorescence.
INTRODUCTION
The development of methods to analyze proteins dates back to the advent of ultraviolet (UV) spectroscopy approach when Warburg and Christian quantified each protein based upon its intrinsic extinction coefficient. 1 Protein quantitation methods that utilized colorimetric analysis then evolved beginning with the biuret procedure and followed by Lowry et al., in the 1950s.2,3 Since then, a steady stream of manually performed protein quantitation methods have appeared that offer lower limits of detection and greater linearity.2,3Included in this group are spectroscopic procedures such as the Bradford dye-binding technique and the bicinchonic acid (BCA) procedure, as well as fluorescent assays such as Fluorescamine binding. 3 While none were initially considered suitable for automation, successful adaptations occurred as illustrated by Moore et al., that described the use of an Autoanalyzer for protein quantitation using the Lowry procedure. 4 Although it yielded more reproducible results than those obtained by manual means, the throughput of this automated Lowry method was low (< 60 samples/hour) and it required a dedicated analyzer. Other undesirable characteristics of early protein quantitation methods included use of caustic agents or complex protocols with multiple reagent additions or incubations.
Recently, a rapidly expanding need for automated protein quantitation has developed in the drug discovery arena based upon high throughput screening. This has stimulated a corresponding interest in automated protein analysis methods that can accurately and rapidly quantify proteins in large numbers of samples. Of the analytical methods to quantify proteins, fluorescence is especially appealing for its sensitivity and dynamic range. 5 A new method to quantify proteins based upon the binding of a fluorescent dye (NanoOrange®) to proteins has been developed 6 that is simple to perform, rapid, and sensitive. The NanoOrange method is therefore attractive for its potential use on a robotic pipetting station to perform automated protein analysis that could then become integrated with other automated biochemical reactions for drug discovery.
Before adapting the NanoOrange reaction to a commercial robotic pipetting station, we examined its reaction protocol and noted the presence of a heating step (e.g., 10 minutes 90–96°C). This need prompted us to further characterize the NanoOrange reaction for its dependence upon temperature and timing as well as examining the use of alternative means of heating (e.g., microwave oven) to potentially accelerate the reaction's completion. Finally, we also wished to evaluate the possible variability of the reaction to changes in protein sequence.
MATERIALS AND METHODS
EQUIPMENT
Dry-bath incubator (#11–718–2), mercury thermometer (#15–160–22), microplate block (#11–718–32), black 96-well flat bottom microplates (#07–200–590), digital thermometer with dual “K” thermisters (#15–078–3A), 1.5 mL flat-top microcentrifuge tubes (#05–408–14) and all chemicals unless otherwise specified were purchased from Fisher Scientific (Pittsburgh, PA). Type I water was used for all reagent preparation. Purified proteins were obtained from the protein molecular weight kit (#MW-SDS-200) from Sigma Chemical (St. Louis, MO) while the NanoOrange protein quantitation reagent (N-6666) was purchased from Molecular Probes (Eugene, OR). An Emerson microwave (model MT3090), with an output frequency of 2.450 GHz and a rating of 1,250 watts was used for all microwave testing. A research spectrophotometer (DU-7, Beckman-Coulter, Brea, CA) was used to quantify purified proteins based upon their published ultraviolet extinction coefficients. 7 All fluorescence measurements were obtained from a microplate multimode scanner, “ULTRA” provided by TECAN Instruments US (Raleigh, NC).
TEMPERATURE CALIBRATION OF THERMISTERS
To validate the accuracy of the thermisters, both were placed in a beaker of visibly boiling distilled water along with a separate mercury thermometer to serve as a temperature reference. The mercury thermometer was given three minutes to equilibrate while the digital thermisters (test and reference) were read after thirty seconds.
CALIBRATION OF DRY-BATH INCUBATOR
The dry-bath incubator plus microplate heating blocks were calibrated according to temperature readings observed using a mercury thermometer placed near the right-hand edge to measure the block temperature. The temperature of the heating blocks was recorded after five minutes equilibration at selected temperatures spanning 45 − 95 °C (results not shown).
TEMPERATURE RESPONSE OF MICROPLATE WELLS USING THE HEATED DRY BLOCK
Using the equipment described above, the temperature response of a filled microplate (250 μL deionized water/well) was analyzed with the digital thermometer at equilibrated hot block temperatures of 45, 55, 65, 75, and 95°C. After the hot block reached thermal equilibration at each specified temperature, a filled microplate was placed on the block's surface. Temperatures were recorded from two different microplate wells, B1 (outer), and E7 (inner), every two minutes for twenty minutes.
TEMPERATURE RESPONSE OF MICROPLATE WELLS USING THE MICROWAVE OVEN
A microplate was filled with 250 μL dIH2O/well and the temperature response of the microplate heated in the microwave (at full power) was monitored in three different wells, B1, E5 and H9, using the digital thermometer. The microplate was heated at full power three times in thirty-seconds intervals.
CREATING A PROTEIN STANDARD CURVE USING A HOT BLOCK
The influence of increasing the incubation (or reaction) temperature upon the NanoOrange reaction with BSA was studied at 45, 55, 65, 75, and 85 °C. The protein dilutions were prepared in 1.5mL microcentrifuge tubes and mixed with 1.0 mL of 1X Working NanoOrange, then 250 μL aliquots were pipetted into wells at three column positions (#1, 5, and 9) of the microplate. 8 Triplicate aliquots were pipetted into microplate column one to determine if an ‘edge effect’ was present, column five, where the heat is the most intense and an intermediate position (column nine) to examine the consistency of the heating. One of five loaded microplates was then incubated for 20 minutes at each of five pre-equilibrated block temperatures and the reaction's fluorescence quantified (excitation wavelength = 485 nm, emission wavelength = 595 nm.) using the Tecan ULTRA. Multimode microplate scanner.
CREATING A PROTEIN STANDARD CURVE USING A MICROWAVE
A working solution of NanoOrange (‘1X Working NanoOrange’) was prepared by diluting stock NanoOrange protein quantitation reagent 500-fold into 1X protein quantitation diluent, then protected from light until used for protein analysis. Approximately 40 − 80 mL of Working NanoOrange working reagent usually was prepared and the unused portion discarded at the end of each day. Working NanoOrange was used as a diluent to create a series of protein standards diluted from a stock solution of 2 mg/mL purified bovine serum albumin (BSA, Molecular Probes). Concentration of these standards ranged from 0.1 μg/mL to 10 μg/mL. The series of protein standards diluted with 1X Working NanoOrange reagent were then delivered in 250 μL aliquots into selected columns of wells (#1, 5, and 9) of a microplate. Each protein standard concentration was prepared in triplicate. The microplate was placed on a glass rotating plate in the microwave and heated at full power for three, thirty-second intervals with a thirty-second break between each heating cycle. Total fluorescence of the NanoOrange reaction in each well was quantified using Tecan ULTRA microplate scanner.
DETERMINATION OF PROTEIN-TO-PROTEIN VARIATION OF NANOORANGE REAGENT USING THE HOT BLOCK
To determine if a sequence-based variation occurred when NanoOrange reacted with different proteins, its reaction with BSA was compared with carbonic anhydrase (CA, bovine) and ovalbumin (OVA, chick). The latter two proteins were contained in the SDS Molecular Weight standards kit purchased from Sigma Chemical Co, St. Louis, MO. Stock solutions of CA and OVA were prepared by dissolution in 10 mM TRIS (pH 8.0) and dIH2O. Concentration of protein stock solutions were determined using published UV extinction coefficients 7 and the corresponding UV absorbance quantified using the DU-7 spectrophotometer. These stock protein solutions were diluted to create concentration series and reacted with NanoOrange using the procedure previously described.
RESULTS
HEATING RESPONSE OF MICROPLATE
Accuracy of temperature measured by mercury thermometer vs. digital thermometer with “K” thermisters demonstrated temperatures less than 1°C different between the two devices. The mercury thermometer read within 2°C of expected for boiling water at this altitude.
Heating Block: Temperatures in the outside wells of a microplate were consistently lower than those located in the center of the microplate (“edge effect”). Results in Figure 1 illustrate that this temperature difference gradually increased from 4°C at 55°C block temperature to almost 7°C at 95°C block temperature. Moreover, temperatures within the microplate wells never achieved the block temperature but always stabilized to lower equilibrated values within 15 minutes (time results not shown) and never exceeded 65°C. At 95°C block temperature, the microplate physically warped while being heated during the incubation period. As the plate reader could not accurately read the warped microplate, fluorescent results were not recorded from microplates incubated at 95°C.
Temperature response of microplate heated with hot block. 96 well microplate filled with 250 μL dIH2O/well positioned on hot block at indicated temperature. Temperatures displayed are from 15 minutes reading. Temperatures after 15 minutes did not change (results not shown).
Microwave: By contrast, temperatures in the microplate that was heated using a microwave show relatively uniform heating across the entire microplate as illustrated by results in Table 1. Temperatures in the edge wells (B1 and H9) were about the same as the interior well (E5). However, no well temperatures ever exceeded 50°C during the heating phase.
Temperature response of Microplate Heated in a Microwave Oven. Note: 96 well microplate (filled with 250 μL dIH2O)/well) and heated for three 30 second cycles at full power (1250 watts). Well locations (B1,E5, H9) refer to positional coding denoted on rim of a standard 96 well microplate
EFFECTS OF HEATING BLOCK FORMAT ON PROTEIN STANDARD CURVE
As seen in Figure 2, the family of standard curves created using BSA and NanoOrange reagent for the microplate heated on dry block are curvilinear and resemble the one displayed by the manufacturer. 8 In particular, the results of the microplate block testing show a marked increase in sensitivity from 45°C to 65°C. From 65°C to 85°C, however, the shape and intensity of the higher temperature response curves appear to be very similar and overlay each other in shape and fluorescence value (Figure 2).
Response of NanoOrange binding reaction to bovine serum albumin (BSA) after heating at selected temperatures. The fluorescence values at each BSA concentration are the mean ± SE for triplicate determinations. Note the response curves overlay each other at 65, 75, and 85 °C.
ANALYSIS OF NANOORANGE REAGENT OF THREE PROTEINS USING MICROPLATE:HOT BLOCK METHOD
Initial tests of two lyophilized proteins (BSA and OVA) that were dissolved in TRIS (pH 8) solution yielded standard curves with inconsistent fluorescent results (results not shown). The third protein, carbonic anhydrase (CA), produced virtually no fluorescence at higher protein concentrations (> 6 ug/mL) when initially dissolved in TRIS (pH 8) (results not shown). Purified CA was then dissolved in distilled water. The fluorescence of the CA standard curve prepared in distilled water matched standard curves of the BSA and OVA and was similar to earlier results of BSA standard (Figure 3). The graph also shows that the microwave method of BSA yields much lower fluorescence than the three proteins assayed in the microplate directly using hot block for heating. The initial slope (range: 0 to 0.5 μg/mL) of the protein concentration curves from the three tested proteins was greater in comparison to that of BSA analyzed in the microwave method (Figure 3).
Response curves for three proteins using NanoOrange reaction. To p three curves are for NanoOrange: protein reaction heated at 65 °C on a hot block for 20 minutes. The lowest curve is the NanoOrange: BSA response curve when heated in a microwave oven. The fluorescence values at each protein concentration are the mean ± SE for triplicate determinations. CA = carbonic anhydrase, BSA = bovine serum albumin, OVA = ovalbumin, BSA (mcrw) = BSA assayed using microwave heating.
DISCUSSION
High throughput automation represents a recent technology aimed at improving laboratory efficiency sought by the pharmaceutical industry. The ability to automate protein quantitation would be beneficial for the increase in analytical throughput. Ease of use and high sensitivity make the NanoOrange method a prime candidate for automation and high throughput screening. However, the assay presents challenges as well. First, the protocol 8 suggests a heated incubation period (e.g., 10 minutes) at elevated temperatures (e.g., 90 − 96 °C). Second, both stock NanoOrange reagent and 1X Working NanoOrange are light sensitive. In the later case, this may prohibit use of the reagent destined for long-term (four to six hours) storage on the deck of a pipetting station. The first constraint prompted us to further investigate the reagent's need for heating while the second could be addressed using a pierceable light shroud placed over the working reagent.
According to the manufacturer's instructions, the NanoOrange assay must be heated to a temperature of 95 °C for 10 minutes after the protein sample has been added. 8 While heating can be accomplished in a variety of ways, several recent publications also demonstrate that incubating the NanoOrange reagent with protein sample at room temperature9–11 also generated an effective fluorescent signal. Moreover, Liu at al., 10 demonstrated that the room temperature reaction between NanoOrange and BSA had a reaction half time of ∼110 msec, so that the reaction was > 99.5 % complete in one second after mixing. Thus extended incubation times at elevated temperatures may not be necessary, although additional studies would be needed to clarify the optimum time necessary. Clearly, shorter incubation times would be beneficial to the automation of the NanoOrange reaction.
To better understand the influence of temperature upon the NanoOrange: protein reaction, we first placed a filled microplate upon a calibrated dry-block heater, allowed it to equilibrate at a series of temperatures (45, 55, 65, 75, and 95°C), then recorded well temperatures every two minutes for twenty minutes. Figure 1 demonstrates that the outer edge heats at a slower rate than that of the inner wells. This ‘edge effect’ may be important in understanding the accuracy of the NanoOrange reaction with proteins as a selected block temperature may not be reached by the outer wells, possibly compromising the results. (Note: The manufacturer's protocol suggests that the reaction mixture be incubated at a 90–96 °C range for ten minutes, but fails to mention the minimum reaction temperature needed.) At a dry block temperature of 95 °C, a microplate with 250 μL distilled water/well reached a temperature of 58 °C in the outer well and 65 °C in the inner well. Thus, the highest temperature the NanoOrange assay achieves in a microplate heated on a dry block is 65 °C, well below the 90 − 96 °C recommended by the manufacturer. 8
Moreover, a close examination of the results in Figure 2 reveals that block temperatures of 65 °C or greater gave no difference in magnitude of fluorescent signal from the standard curves. Outer and inner well temperatures for the block at 65 °C were 42 and 47 °C respectively, which suggests that once the NanoOrange reaction with BSA achieves a temperature of 42 °C, it has reached completion. However, since other proteins may require additional time and increased temperature for complete denaturation, 8 further studies with different proteins are needed to clarify this issue.
An alternative method to heat the NanoOrange assay uses a microwave (method protocol provided by Molecular Probes). Chemical reactions accelerated by microwave technology is a relatively new development that involves effectively heating samples through the use of 2.45 GHz radiofrequency radiation. 12 Recent trends in the use of microwave radiation include facilitating organic chemical synthesis 13 and creating combinatorial libraries. 14 When we followed the microwave method provided by the NanoOrange manufacturer (K. Free, email procedure, Molecular Probes Technical Service, June 15, 2001), well temperatures were 10 °C lower (Table 1) than those observed using the original hot-block protocol (Figure 1). For example, after three cycles (30 seconds) in our microwave, 250 mL of distilled water in a microplate well only reaches 45 − 49 °C. Others have noticed that the field and energy distribution in microwave ovens may be erratic, 12 14 which may explain the difference between our fluorescence reading and those from the hot block.
The shape of the microwave-based NanoOrange curve in Figure 3 displays lower intensity than those derived using the hot block, but still indicates that if a microwave heating protocol can be developed to provide uniform temperature in the microplate, a faster result could be obtained. This approach has been confirmed by a microwave-accelerated BCA protein assay that was finished after one 20 second heating, although the authors used 2.5 mL tubes (not microplates) and included a 100 mL volume of water as a heating ballast. 15
A third method to provide heat for the NanoOrange assay used 1.5 mL microcentrifuge tubes. These tubes were filled with 1 mL of liquid (equivalent to four tests when aliquoted into a microplate) and placed in the dry-block heater. The block was adjusted to 93°C and the results indicate that after six minutes incubation, the liquid in the microcentrifuge tubes reached 88°C (results not shown). This heating method was the only one to achieve a temperature close to the desired value of 90 − 96 °C, but since 1.5 mL tubes cannot be readily manipulated by robotic pipetting workstations, this heating format was not pursued.
Closer examination of Figure 2 reveals that at microplate block temperatures of 45 °C, the NanoOrange reagent yielded a curvilinear graph with lowered sensitivity for protein quantitation, but at 65 °C or above, the protein standard curves are nearly identical. This lower temperature of 65 °C would open the NanoOrange reagent to more applications and is consistent with results from investigators who have used NanoOrange at room temperature.9–11 Further comparison of the hot block's response curves indicate they are similar in shape to the microwave fluorescence response curve, but the latter's fluorescence values are significantly lower. This difference suggests that the two heating procedures are not interchangeable. Finally, NanoOrange's curvilinear response to protein concentration is not unique as this nonlinearity has also been observed previously with the Lowry assay. 2 CBB dye, 16 and BCA assay. 17
Bovine serum albumin (BSA) is commonly used as a standard for a variety of protein quantitation methods. The NanoOrange method also uses BSA as a calibrator and claims to be capable of quantifying proteins at a much lower concentration (microplate = 100 ng/mL) than traditional spectroscopic methods such as Lowry, Bradford, and BCA. 3 While two of these colorimetric methods (Lowry and BCA) exploit a chemical reactivity of proteins for quantitation, the Bradford method uses the binding of Coomassie Brilliant Blue G-250 (CBB) to proteins to create the chromophore. Because the Lowry, 2 CBB, 3 and BCA 17 methods are sensitive to protein sequence, all proteins do not react within these reagents in equimolar amounts. By contrast, the initial (< 0.5 μg/mL) slopes of the three proteins in Figure 3 indicates that the NanoOrange binds and reacts with these proteins with similar molar binding ratio (Figure 3). Thus our results suggest a BSA standard reacted with NanoOrange would yield an accurate concentration of different proteins regardless of their sequence.
We also discovered that proteins dissolved in 10 mM TRIS (pH 8) displayed distorted concentration response curves with sharp maxima of fluorescence around 5 ug/mL that rapidly descended at higher protein concentrations (results not shown). Significant time-based alteration in NanoOrange's reaction with protein in the presence of TRIS has been described by others. 11 Caution should thus be excercised when using NanoOrange to analyze proteins that have been dissolved in 10 mM TRIS.
SUMMARY
Our results and those of others reveal that NanoOrange rapidly reacts with proteins with heat 8 or at room temperature.9–11 However, for generic protein quantitation using NanoOrange, heating appears to facilitate the dye's binding reaction to proteins. We observed that the maximum fluorescence signal is attained when the well contents achieve temperatures of 65°C or greater in fifteen minutes.
While use of microwaves may provide a quicker heating within a microplate, a small microwave compatible of being positioned on an automated pipetting platform is presently unavailable. Moreover, there remains the need for a microwave of at least 600 watts power to adequately heat the contents of a microplate. By contrast, a hot block adjustable to 65–85°C that can hold a microplate could be positioned on the deck of a liquid handling robot, thus permitting the automated quantitation of proteins with NanoOrange.
A potential diagnostic application of NanoOrange using capillary electrophoresis to analyze human serum albumin may also accelerate its development as a rapid means to quantify proteins. 11 Successful adaptations of the NanoOrange method to automated platforms will thus likely lead to more rapid procedures for both diagnostic and research laboratories.