Thermo Scientific NanoDrop Products
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Desjardins, P., Hansen, J. B., Allen, M. Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer. J. Vis. Exp. (33), e1610, doi:10.3791/1610 (2009).
Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 μL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.
I. The NanoDrop 2000c Spectrophotometer
NanoDrop technology is based on an innovative sample retention system that uses the surface tension to hold and measure microvolume samples between two optical pedestals without the use of cuvettes or capillaries. The NanoDrop 2000c spectrophotometer uses this technology to quickly and easily measure 0.5-2 μL droplets of proteins, DNA, RNA, and other biomolecules. This capability has become increasingly important as molecular techniques continually evolve to use smaller amounts of material for analysis. The microvolume spectrophotometer ideal for conditions in which sample is limited. However, the ease-of-use, fast measurement cycle and, and extensive concentration range also make the spectrophotometer suitable when ample amounts of sample are available. The measurement cycle is also greatly reduced, helping scientists increase efficiency throughout their workflows.
The microsample is placed directly on top of the detection surface and a liquid column is created between the ends of the optical fibers by surface tension. This liquid column forms a vertical optical path. A xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light that passes through the sample.
Removing traditional containment devices such as cuvettes from the system has several advantages: very small amounts of sample are needed for measurement, cleanup simply requires wiping the optical surfaces with a laboratory wipe, and the path length can be changed in real time during the measurement.
The NanoDrop 2000c determines the optimal path length automatically (1 mm to 0.05 mm), providing the most extensive range of possible protein concentration measurements without dilutions. By shortening the path length, higher concentrations of protein can be measured. This effectively removes the need to perform dilutions for most protein samples. For example, the NanoDrop 2000c can measure BSA concentrations as high as 400 mg/mL.
The NanoDrop 2000c Spectrophotometer is a full spectrum spectrophotometer for measuring the absorbance of DNA, RNA, proteins, and other biomolecules. This video protocol will focus on the measurement of proteins.
II. Microvolume Protein Concentration Determination Using A280 Absorbance Measurements
a. Principle of A280 Measurements
The Protein A280 method is applicable to purified proteins that contain Tryptophan, Tyrosine, Phenylalanine residues or Cysteine-Cysteine disulphide bonds and exhibit absorbance at 280 nm. This method uses the A280 absorbance value in combination with either the mass extinction coefficient or the molar extinction coefficient to calculate the concentration of the purified protein. The advantage of direct A280 measurements is that the generation of a standard curve is not required to determine protein concentration. If the sample is an uncharacterized protein solution, cell lysate, or crude protein extract, then using one of the pre-configured colorimetric methods available on the NanoDrop 2000/2000c, such as BCA, Pierce 660 nm, Bradford, and Lowry assays, is recommended.
b. Microvolume Protein A280 Measurements - Startup
c. Microvolume Protein A280 Measurements - Blanking
d. Microvolume Protein A280 Measurements Measuring
Important considerations: The homogeneity of the sample is extremely important since such a small volume is being measured. To ensure that samples are homogenous, gently but thoroughly mix the samples immediately prior to taking an aliquot for measurement. Avoid introducing bubbles when mixing and pipetting.
Due to the variability in surface tension between different proteins, we recommend loading 2 μL of samples to ensure proper column formation. Always use low retention pipette tips. To load a sample, touch the pipette tip to the lower optical pedestal surface while expelling the solution to prevent the solution from adhering to the outside of the pipette tip. Expel less than the full amount of sample to prevent blowout and introduction of bubbles in the sample.
e. Microvolume Protein A280 Measurements Cleaning
An ordinary, lint-free, laboratory wipe is often sufficient for cleaning the optical pedestals between measurements.
f. Making Protein A280 Measurements Reconditioning
Solutions and reagents containing surfactants may uncondition the measurement pedestal surfaces over time, preventing the sample liquid column to form. A flattening of the droplet on the lower pedestal is indicative of the optical surface becoming unconditioned. If the surface properties have been compromised, reconditioning the pedestals is important to ensure sample column formation. If this occurs, buff the optical surfaces vigorously using laboratory wipe or use NanoDrop Pedestal Reconditioning Compound (PR-1) as directed.
III. Microvolume Protein Concentration Determination Using Colorimetric Assays
a. Principle of colorimetric detection
The NanoDrop 2000c spectrophotometer can also be used to measure uncharacterized protein solutions, cell lysates, and crude protein extracts using colorimetric assays.
Colorimetric methods are indirect methods that involve interaction of a dye with the protein component of the sample to produce a new complex that absorbs light in the visible wavelength range.
The NanoDrop 2000c spectrophotometer has several pre-configured colorimetric assays including BCA, Pierce 660, Bradford, and Lowry methods. The BCA assay will be demonstrated as an example colorimetric assay using a microvolume spectrophotometer. (screenshot)
The BCA (Bicinchoninic Acid) assay is a common colorimetric method often used for dilute protein solutions and proteins in the presence of components that have significant UV (280 nm) absorbance. Unlike the Protein A280 method, the Protein BCA method requires that a standard curve be generated before sample protein concentrations can be measured.
The method uses bicinchoninic acid (BCA) as the detection reagent. The Cu-BCA chelate formed in the presence of protein is measured at 562 nm and normalized at 750 nm.
b. Microvolume BCA Assay Measurements BCA Assay Preparation
c. Microvolume BCA Assay Measurements Generating Standard Curve
d. Microvolume BCA Assay Measurements 2 μL Protein Measurements
e. Microvolume BCA Assay Measurements Cleaning and Reconditioning
Perform cleaning and reconditioning as described in the direct A280 absorbance measurements.
Microvolume protein concentration determination is performed by either a direct A280 measurement or an indirect colorimetric assay. The A280 measurement example determines protein concentration based on the extinction coefficient of the protein of interest. The BCA colorimetric assay example determines protein concentration based of a standard curve of known protein concentrations.
Microvolume quantitation uses the intrinsic surface tension properties of a sample to form a liquid column between two measurement surfaces. The absence of a containment device, allows the path length to change in real time, essentially eliminating the need to perform dilutions. This capability dramatically increases the dynamic range of protein concentrations as well as the speed of measurement. By using minimal amounts of sample, a large number of samples can be analyzed quickly and accurately, allowing scientists to take more measurements and achieve better quality control. In addition, and if necessary, precious samples can be recovered after taking a measurement. When such small volumes are being measured, sample homogeneity is extremely important to avoid sampling errors. Low retention pipette tips should always be used for loading samples and the tips should be changed between sample replicates. The pedestal surfaces must be properly cleaned and conditioned to ensure the most accurate results. Finally, the microvolume spectrophotometer is ideal when sample is limited, however, the ease-of-use, speed, and extensive dynamic range of the spectrometer make it suitable when sample is plentiful.
The authors are employees of Thermo Fisher Scientific that produces reagents and tools used in this article.
|NanoDrop 2000c Spectrophotometer||Thermo Fisher Scientific, Inc.|
|Pierce BCA Protein Assay Kit||Thermo Fisher Scientific, Inc.||23250||Reducing agent compatible|
|PR-1 Reconditioning Kit||Thermo Fisher Scientific, Inc.|