Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

NanoDrop technology combines fiber optics and surface tension to hold and measure small amounts of samples such as nucleic acids and proteins. A small droplet of sample is pipetted directly onto the lower pedestal. An upper optical pedestal then engages the sample to form a column.

The pedestal automatically moves to adjust for optimal path length. Shortening the path length eliminates the need to perform dilutions for most protein samples, measurement occurs in seconds and vis spectrum results are displayed. Hi, I'm Lynn Kalhorn at the NanoDrop Laboratories of Thermo Fisher Scientific.

Today we will show a procedure for determining protein concentration using an innovative micro volume spectrophotometer. The NanoDrop 2000 C.The NanoDrop 2000 C spectrophotometer uses an innovative sample retention system that relies on the surface tension of liquids to hold and measure micro volume samples between two optical pedestals without the use of cuvettes or capillaries. The micros sample 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. One to two microliters cleanup simply requires wiping the optical surfaces with a laboratory wipe and the path length automatically changes in real time during the measurement. In addition, shortening the path length enables the measurement of higher concentrations, which effectively removes the need to perform dilution for most protein samples.

In cases in which a vet is warranted, such as kinetic studies, the NanoDrop 2000 C provides a vete option in the same spectrophotometer. For this protocol, we will use the micro volume pedestal option to determine protein concentration. The protein A two 80 method is applicable to purified proteins that contain tryptophan tyrosine and phenyl alanine residues or cysteine cysteine diss sulfide bonds and exhibit absorbence at 280 nanometers.

To begin this procedure, select the protein A two 80 application from the main menu. If the wavelength verification window appears, ensure the arm is down, select the type of sample to be measured from the dropdown list. The default setting is recommended for most unknown protein mixtures in which one ABSORBENCE equals one milligram per milliliter.

If measuring a previously characterized purified protein, then either the mass extinction coefficient or molar extinction coefficient may be entered. To determine the protein concentration more precisely, choose the concentration units from the dropdown list adjacent to the color coded box. Next, establish a blank.

Using an appropriate buffer, it is important to use the buffer in which the protein is suspended. The buffer used should be of the same pH and a similar ionic strength as the sample solution. Due to the variability in surface tension between different proteins, we recommend loading two microliters of samples to ensure proper column formation.

To load touch the low retention pipette tip to the bottom 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 into the sample. Once the sample is loaded, lower the arm, click blank. When blanking is complete, wipe the blank solution from the lower and upper pedestals.

Using a dry laboratory wipe prior to taking an aliquot for measurement, it is important to ensure that the sample is homogenous. Mix the sample gently but thoroughly by gentle vortexing using a mid-level RPM to avoid introducing microbubbles into the fluid. Enter a sample ID in the appropriate field.

Load two microliters of the first sample as shown previously for the blank, lower the arm and click measure When measurement is complete, wipe the sample from the lower and upper pedestals. Using a dry laboratory wipe, measure all the other samples in the same manner using a fresh two microliter aliquot of sample for each measurement. An ordinary lint-free laboratory wipe is often sufficient for cleaning the optical pedestals between measurements.

The only solvent that is incompatible and must not be used is hydrofluoric acid solutions and reagents containing surfactants may uncondition the measurement pedestal surfaces over time preventing formation of the sample liquid column. A flattening of the droplet on the lower pedestal is indicative of the optical surface becoming uncondition to recondition. The pedestal surfaces either buff them vigorously using a laboratory wipe or the preferred method is to use the NanoDrop pedestal reconditioning compound PR one as directed.

If the sample is an uncharacterized protein solution, cell lysate or crude protein extract, we recommend using one of the pre-configured colormetric methods available on the NanoDrop 2000 C.These colormetric methods include BCAP six 60 Bradford and Lowy assays. The BCA assay will be demonstrated here. The required reagents for the BCA assay are contained in the thermos scientific pierce reducing agent compatible kit BCA, reagent A, B, C, A, reagent B, and bovine serum albumin standards.

We will be using pierce's pre-diluted albumin set as our standards refer to the pierce BCA literature to determine which assay standards are appropriate for your circumstance. To prepare the reagents first equilibrate all unknown proteins and protein standards to room temperature and mix thoroughly by gentle vortexing. Use a mid-level RPM to avoid introducing microbubbles into the fluid.

Since bubbles may negatively affect the readings. Prepare enough fresh working reagent for the standards and samples to be measured using a 50 to one ratio of reagent A to B.In this example, we will prepare three milliliters of working reagent to cover the reference five standards and six samples. The regular BCA assay covers a wider dynamic range and uses a 20 to one reagent to protein ratio, whereas the micro BCA assay is more sensitive and has a narrower dynamic range using a one to one reagent to protein ratio.

Because we are performing the regular BCA assay, we will prepare a 20 to one ratio of working reagent to sample. Add 190 microliters of working reagent to each tube with enough tubes to cover all samples and standards. Next, add 10 microliters of standard or sample to each of the tubes containing reagent.

Mix well by gentle vortexing. Incubate the tubes at 37 degrees Celsius for 30 minutes. After the incubation, allow the reactions to equilibrate to room temperature for about 10 minutes.

Unlike the protein A two 80 method, the protein BCA method requires that a standard curve be generated before sample protein concentrations can be measured. To generate a standard curve, start by selecting the protein BCA method from the main menu. If the wavelength verification window appears, ensure the arm is down.

Enter the values for each standard concentration in the right pane table. The software allows for the reference and up to seven additional standards. The reference and or standards can be measured in replicates.

Establish a blank using the appropriate buffer. The blank for color metric assays is generally deionized. Water pipette two microliters of water onto the bottom pedestal and lower the arm.

Click blank. Only one blank is necessary to cover all subsequent measurements of the reference and standards. Establish the reference by pipetting a two microliter Eloqua of only working reagent and buffer with no protein onto the lower pedestal.

Lower the arm and click measure. Under the standards tab, highlight the desired standard and pipette two microliters of the desired standard onto the lower pedestal. Lower the arm and click measure.

Repeat the process for all standards. To view the standard curve, click view standard curve. After all the standards have been measured, click on the samples button.

Enter the sample ID load two microliters of sample onto the lower pedestal and click measure. A fresh two microliter aliquot of sample should be used for each measurement. When measurement is complete, wipe the sample from the lower and upper pedestals using a dry laboratory wipe.

The result is then obtained as the final concentration of the sample is automatically calculated using the standard curve. We've just shown two ways of determining protein concentration, either by direct a two 80 measurement or by an indirect color metric method each using the micro volume NanoDrop 2000 C spectrophotometer. That's all.

Thanks for watching.