Name: microvolume protein concentration determination using the nanodrop 2000c spectrophotometer
Uploaded: 2009-11-04T16:00:00.000+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.

<table><tbody><tr><td>NanoDrop 2000c Spectrophotometer</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr><tr><td>Laboratory wipes</td><td></td><td></td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Fisher Scientific, Inc.</td><td>23250</td><td>Reducing agent compatible</td></tr><tr><td>PR-1 Reconditioning Kit</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr></tbody></table>

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

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Video: Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Cancer Research

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Chemistry

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Bioengineering

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

Engineering

Expanding the Mass Photometry Analysis Concentration Range with Microfluidics

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

Author Spotlight: Characterization of Low-Affinity Protein Interactions in Solution Using MassFluidix Technology

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

Biochemistry

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 ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...