Name: microvolume protein concentration determination using the nanodrop 2000c spectrophotometer
Uploaded: 2009-11-04T16:00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer at JoVE.com

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 &mu;L 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
<ol>
 <li>Select the Protein A280 application from the main menu. </li>
 <li>Select the type of sample to be measured from the drop-down list. The default setting is recommended for most unknown protein mixtures in which 1 Abs = 1 mg/mL. If measuring a previously characterized purified protein, then either the mass extinction coefficient or molar extinction coefficient and molecular weight may be entered to determine the protein concentration more precisely. </li>
 <li>Choose the concentration units from the drop-down list.</li>
</ol>
c. Microvolume Protein A280 Measurements - Blanking
<ol>
 <li>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 the same pH and of a similar ionic strength as the sample solution. Note: RIPA buffers contribute too much absorbance at 280 nm and are therefore incompatible with direct A280 protein measurements. For proteins suspended in a RIPA buffer, it is recommended protein concentration be determined using a RIPA buffer compatible colorimetric assay.</li>
 <li>Pipette 2 &mu;L of the appropriate blanking solution onto the bottom pedestal, lower the arm and click Blank. </li>
 <li>Wipe the blank solution from the lower and upper pedestals using a dry laboratory wipe.</li>
</ol>
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 &mu;L 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. 
<ol>
 <li>Enter a sample ID in the appropriate field, load 2 &mu;L of the first sample as described for the blank and click Measure. A fresh 2&mu;L aliquot of sample should be used for each measurement. </li>
 <li>Remove the sample from the lower and upper pedestals using a dry laboratory wipe.</li>
</ol>
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
<ol>
<li>The required reagents are contained in the reducing agent compatible kit (Thermo Scientific Pierce cat. no. 23250): BCA reagent A, BCA reagent B, Compatibility reagent, Reconstitution buffer, and Albumin (BSA) standards.</li>
<li>Equilibrate all unknown proteins and protein standards to room temperature and mix thoroughly.</li>
<li>Prepare enough fresh working reagent for the standards and samples to be measured using a 50:1 ratio of reagent A:B</li>
<li>For the micro assay, use a 1:1 ratio of working reagent to sample. Add 10 &mu;L of working reagent to each tube with enough tubes to cover all samples and standards. For the high-range assay, use a 20:1 ratio of working reagent to sample. Add 190 &mu;L of working reagent to each tube with enough tubes to cover all samples and standards. Refer to the Pierce BCA kit literature to determine which ratio is suitable for your samples.</li>
<li>Add 10 &mu;L of standard or sample to each tube containing reagent. Mix well by gently vortexing.</li>
<li>Incubate the tubes at 37 &deg;C for 30 minutes.</li>
<li>Allow the reactions to equilibrate to room temperature (~10 minutes).</li>
</ol>
c. Microvolume BCA Assay Measurements Generating Standard Curve
<ol>
 <li>Select the Protein BCA method from the main menu. If the wavelength verification window appears, ensure the arm is down. </li>
 <li>Enter the values for each standard concentration in the right pane table. The software allows for the reference and up to 7 additional standards. The Reference and/or standards should be measured in replicates. 
 Note: The minimum requirement for standard curve generation is the measurement of two standards or the measurement of the zero reference and at least one standard. It is recommended that additional standards be included as necessary to cover the expected assay concentration range.</li>
 <li>Establish a blank. The blank for colorimetric assays is generally deionized H2O.</li>
 <li>Pipette 2 &mu;L of dH2O onto the bottom pedestal, lower the arm and click Blank. Only one blank is necessary to cover all subsequent measurements of the reference and standards.</li>
 <li>Establish the reference by pipetting a 2 &mu;L aliquot containing only working reagent and buffer with no protein onto the lower pedestal. Lower the arm and click Measure.</li>
 <li>Under the Standards tab, highlight the desired standard and pipette 2 &mu;L of the desired standard onto the lower pedestal. Lower the arm and click Measure. Repeat the process for all standards. Be sure to measure all standards prior to measuring samples. To view standard curve click View Standard Curve. </li>
</ol>
d. Microvolume BCA Assay Measurements 2 &mu;L Protein Measurements
<ol>
 <li>After all of the Standards have been measured, click on the Samples button. Enter the sample ID. Load 2 &mu;L of sample onto the lower pedestal and click Measure. A fresh 2 &mu;L aliquot of sample should be used for each measurement.</li>
 <li>Wipe the sample from the lower and upper pedestals using a dry laboratory wipe.</li>
</ol>
e. Microvolume BCA Assay Measurements Cleaning and Reconditioning
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.

<ol>
	<li>Aitken, A., Learmonth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+determination+by+UV+absorption.">Protein determination by UV absorption.</a> , (1996).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J. Biol. Chem. 193, 265-275 (1951).</li><li>Reisner, A. H., Nemes, P., Bucholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Coomassie+Brilliant+Blue+G250+perchloric+acid+solution+for+staining+in+electrophoresis+and+isoelectric+focusing+on+polyacrilamide+gels.">Use of Coomassie Brilliant Blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrilamide gels.</a> Anal. Biochem. 64, 509-516 (1975).</li><li>Simonian, M. H., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+colorimetric+determination+of+protein+concentration.">Spectrophotometric and colorimetric determination of protein concentration.</a> Curr. Protoc. Mol. Biol. 10:1A.17. , (2006).</li><li>Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, F. u. j. i. m. o. t. o., Goeke, E. K., Olson, N. M., J, B., Klenk, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Meth. Enzymol. 182, 50-68 (1990).</li></ol>

The authors are employees of Thermo Fisher Scientific that produces reagents and tools used in this article.

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.

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<td>NanoDrop 2000c Spectrophotometer</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Laboratory wipes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Pierce BCA Protein Assay Kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>23250</td>
<td>Reducing agent compatible</td>
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<td>PR-1 Reconditioning Kit</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
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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 &mu;L. 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.

Watch this Scientific Journal Video about Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer at JoVE.com

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

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microvolume-protein-concentration-determination-using-nanodrop-2000c

Research

JoVE Journal

Biology

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...