Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

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.


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. Perform cleaning and reconditioning as described in the direct A280 absorbance measurements.

Representative Results:
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.

Discussion
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.