Quantify Your Proteins With Nanodrop Instruments
Thermo Scientific NanoDrop UV-Vis spectrophotometers support protein sample quantification with applications for direct A280, A205 and colorimetric assays . There are several things to consider when deciding which method to use to quantify your protein samples using a NanoDrop UV-Vis spectrophotometer.
Is your sample a purified protein? Purified protein samples can be accurately measured using direct absorbance at 280 nm. Absorbance at 280 nm is mostly due to the aromatic chains on the amino acids Tryptophan and Tyrosine . Protein A280 is the most popular quantification method because it is fast and simple, requires no reagents or standard curves, and consumes very little sample. Using the absorbance at 280nm , protein concentration is calculated using the Beer-Lambert equation A280 = c * * b . Each pure protein has a unique extinction coefficient. For accurate results, the correct protein extinction coefficient must be entered or the closest Sample Type must be selected. The NanoDrop One Protein Editor feature allows you to save the extinction coefficients of specific proteins so that you can customize your Sample Type options.
FIG 1. Protein BCA data on the NanoDrop One
FIG 2. Fluorescein labeled IgG measured with Proteins and Labels application on the NanoDrop One
The Bicinchoninic Acid Assay
How it works
The BCA assay is another colorimetric assay like the Bradford assay. It makes use of the biuret reaction, in which the protein backbone chelates Cu2+ ions and reduces them to Cu1+ ions.
The Cu1+ ions then react with BCA to form a purple-colored product that absorbs at 562 nm. The procedure is similar to that of the Bradford assay, in which you create a standard curve based on a series of known protein standards.
Because the peptide backbone is involved in the reaction, the BCA assay is less affected by differences in the amino acid composition of your protein. However, the reaction is influenced by cysteine, tyrosine, and tryptophan residues.
The reagent is not sensitive to detergents and denaturants, so its okay to have those in your buffer.
The presence of reducing agents in your buffer can interfere with the dye, but there are reducing agent-compatible dyes available.
The reaction takes some time to proceed. Usually, the samples are incubated at 37°C for 15-30 min. Also, as in the Bradford assay, you determine your protein concentration by creating a standard curve from a known, standard protein. So again, if your protein doesnt interact with the dye in a similar way as the standard protein, your concentration could be off.
How Do You Calculate Protein Concentration From A280
4.2/5A280DETERMINE PROTEIN CONCENTRATIONprotein concentrationabsorbance280absorbanceprotein
Concentration = Absorbance at 280 nm divided by path length Pure protein of known absorbance coefficient. Use the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is used.
Furthermore, how do you calculate protein concentration from Nanodrop? To measure the protein concentration at 280 nm press the “Protein A280″ button. The programme will ask to place 2 l of deionized water on the lower sample pedestal and then press OK. Wipe the water from BOTH upper and lower sample pedestals with a paper tissue and place 4 l buffer on the lower one.
Similarly, how do you calculate protein concentration?
The concentration of Protein in solution can be determined by substituting the molecular weight, extinction coefficient and max into a derived form of the Beer-Lambert Law. A substance’s max is the wavelength at which it experiences the strongest absorbance. For Protein, this wavelength is 280 nm.
How do you find concentration from absorbance?
The equation should be in y=mx + b form. So if you substract your y-intercept from the absorbance and divide by the slope, you are finding the concentration of your sample.
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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.
The BCA assay is a common colorimetric method often used for dilute protein solutions and proteins in the presence of components that have significant UV 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 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
The required reagents are contained in the reducing agent compatible kit : BCA reagent A, BCA reagent B, Compatibility reagent, Reconstitution buffer, and Albumin standards.
Equilibrate all unknown proteins and protein standards to room temperature and mix thoroughly.
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
Select the Protein A280 application from the main menu.
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.
Choose the concentration units from the drop-down list.
c. Microvolume Protein A280 Measurements – Blanking
d. Microvolume Protein A280 Measurements Measuring
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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 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.
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.
Use Of The Nanodrop Spectrophotometer
Open the software of the NanoDrop by double clicking at the icon “ND-1000 V.3.2.1” on the desktop.
To measure the protein concentration at 280 nm press the “Protein A280” button. The programme will ask to place 2 l of deionized water on the lower sample pedestal and then press OK.
If you want a wavelength other than 280 nm or a wavelength scan then you can select “UV-Vis” from the main menu. You can perform a wavelength scan from 220 nm to 750 nm.
- The handling, blanking and measuring is as above.
- In the output you will get the spectrum from 220 nm to 750 nm and you can move the two cursors to the wavelength of your choice or just type it in the two “1” and “2” boxes. Keep in mind though that these are 1 mm path length absorbances and if you want to transform it to the equivalent of a normal spectrophotometer you have to multiply by 10.
Measuring Protein Concentration By Absorbance
The basic approach is to use Beers law to measure the concentration of a protein solution:
A280 is the absorbance of a protein solution at 280 nm.
is the molar extinction coefficient ). This value describes how much 280 nm light a one molar protein solution will absorb over a 1 cm cell.
l is the pathlength in cm. Our spectrophotometer has a pathlength of 1 cm the nanocuvette has a pathlength of 0.1 cm.
c is the protein concentration in molar units.
Using the Eppendorf Spectrophotometer
The apparent absorbance at 280 nm has contributions from the absorption of aromatic residues , as well as from light scattered by aggregate in solution . To measure the scattering contribution, we measure the absorbance at 320 nm and 340 nm where aromatic absorption is minimal, meaning that any signal we observe arises from scatter and then extrapolate back to find the scattering contribution at 280 nm. Scatter increases as ^, so the extrapolation is done on a log/log scale:
This can be estimated from the sequence of the protein by assuming an additive contribution from each Trp, Tyr, Phe, and Cys residue in the protein. Protparam is useful server for doing this calculation.
This can be done, as follows.
Measuring Protein Concentration Bradford Vs Nanodrop
I know that the bradford assay is a very standard way of measuring protein concentration after e.g. a purification. However, in the lab that I work in now they normally only use nano drop at the 280nm wavelength.
How trustworthy is this concentration? Say that I e.g. get 2.5 mg/mL, could I really trust this?
That depends strongly on your protein and how exact you need this concentration. Both tests only do an approximation.
The measurement at 280nm relies on the interaction of aromatic aminoacids with the UV light. If you have a protein with few or no aromatic amino acids, your measurement will be wrong.
The same is true for the Bradford assay. Here you form a complex between Coomassie Brilliant Blue and unpolar and cationic side chains of the amino acids. This test is more precise, since it includes more possible side chains, but is still dependend on the composition of the protein.
More precise is the BCA assay, since it utilizes the reduction of copper ions by the peptide bond of the protein.What you could do is run a BCA assay from your sample and measure the same sample on the Nanodrop at 280nm and compare the results.
The nanodrop should have an option that allows you to input an “extinction coefficient”. This is a measurement of how much one mole of your protein will absorb at 280nm. The nanodrop will use this value to give you an accurate concentration reading.
A few tips:
Some good refs
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Protein Quantification With The Nanophotometer Pearl Protein Uv280 And Colorimetric Based Assays
Dr. Alexis Hofherr, Dr. Andrea HuberRenal Division, Department of Medicine, University Medical Centre Freiburg, Implen GmbH, MunichMay 2011
IntroductionProteins, the dynamic parts of living cells, are involved in nearly all metabolic pathways of cells . The measurement of protein concentration is a frequently used method in life science research. Although there are many assays available, due care should be taken to select the optimum assay for the particular sample type. The decision on which assay to use is typically based on convenience, quantity and purity of protein available, presence of interfering agents, and the accuracy required. In this application note we describe the performance of the NanoPhotometer ® Pearl in terms of linearity and accuracy, applying cuvetteless small volume protein quantification at 280 nm as well as colorimetric cuvette based assays.
Cuvette Based colorimetric Assays These methods are based on the formation of a complex between a specific dye and proteins in solution. Colorimetric assays are more suited for protein mixtures and require an appropriate protein standard.
Technical specifications of the NanoPhotometer® Pearloptical path of the NanoPhotometer® Pearl enables both cuvetteless measurement of protein samples and standard cuvette measurements.
NanoPhotometer® Pearl and Sample Compression Technology are trademarks of Implen GmbH and Implen, Inc.