Protein UV

Proteins, the dynamic parts of living cells, are involved in nearly all metabolic pathways of cells (e.g. enzymatic reactions, immune protection, and cellular response). Protein UV Spectrophotometers are frequently used for the measurement of Protein UV concentrations in life science research. Although there are many assays available, care should be taken to select the optimal assay for the particular sample type. The decision on which assay to use is typically based on convenience, quantity and purity of protein sample available. It is also possible to measure labeled proteins via the absorbance maxima of the dye. Dye concentration and the frequency of incorporation are calculated and displayed automatically.

Measuring Protein UV Concentrations

The Protein UV method exploits the inherent absorbance of proteins at 280 nm in combination with the Beer-Lambert law, where each protein is characterized by a specific extinction coefficient (ε) which can be used to determine total protein concentration of a solution. The intrinsic absorbance of proteins is due to the presence of aromatic amino acids in their structure, primarily tryptophan and tyrosine, as well as cysteine (oxidized cysteine residues in a disulphide bond). The aromatic amino acid residues in a protein containing tryptophan and tyrosine exhibit strong intrinsic absorbance at 280 nm, with a lesser contribution by phenylalanine. Therefore, it is the aromatic amino acid residues which dictate the extinction coefficient at 280 nm for a protein.

The most straightforward method to determine concentration of a purified, homogenous protein with a known extinction coefficient (ε) is by direct measurement of UV280 provided the protein contains no prosthetic groups with strong absorption in the same region. However, for unknown proteins including homogenous protein mixtures, it is possible to make direct A280 measurements using a composite ε value derived from comparison of many proteins, although this will only provide an approximate but close estimate of the true protein concentration.

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The NanoPhotometer®, one of the top Protein UV Spectrophotometers on the market, determines protein concentration by performing calculations based on specific ε values, either pre-programmed in the instrument or entered manually by the user. Extinction coefficient (ε) values at 280 nm vary greatly for different proteins due to their particular aromatic amino acid content.

Fixed ε values are pre-programmed in the software of the Protein UV Spectrophotometer for several proteins. However, if the protein of interest is not included in the pre-programed methods, it is also possible to manually enter the specific ε for the protein of interest using the custom Mol. Ext. Coefficient, custom Ext. Coefficient or custom 1/Ɛ protein factor option. For correct calculation, it is necessary to supply either: a) the molar extinction coefficient (εM in M-1 *cm-1 ) and the molecular weight expressed in molar mass units (g/mol); b) the mass extinction coefficient (ε in l/g*cm) or c) the protein factor 1/Ɛ of the protein.

A common calculator to determine the extinction coefficient of proteins is available here:

To calculate the degree of dye labelling of a protein, the absorbance measured at the wavelength corresponding to the absorbance maximum of the fluorescence dye is used. The corresponding extinction coefficient of the dye is used along with the Beer-Lambert law to determine the dye concentration. Please refer to the manual of the manufacturer of the fluorescence dye used for the labeling of the sample.

Wavelengths for Measuring Protein UV Concentrations

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The Lambert-Beer law, also known as Beer’s law, empirically relates the absorption of light to the properties of the sample. This law states that there is a logarithmic relationship between the transmission of light through a specific sample (T = I/Io with I = outgoing light and Io = incoming light), the molar extinction coefficient for a specific compound (ε), the concentration of the absorbing species in the material (c) and the distance the light travels (d).

The ratio calculation of the absorbance at 260 and 280 nm (260/280 ratio) is monitored to indicate the quality of a protein purification. Different protein structures generally have an influence on the 260/280 ratio. Aromatic amino acids such as tryptophan and tyrosine e.g. absorb at 280 nm, while other amino acids contribute at different wavelengths at which the sample will absorb. Some buffers used in the purification process can also influence the ratio, especially if the buffer contains components that absorb in the same UV spectrum (see graphs below).

A 260/280 ratio less than 0.60 in general is a good indication of whether a protein has been purified with minimal nucleic acid contamination left in the sample. Initial crude lysates may show a much higher value even around 2, this reading will decrease as contaminants are removed during the purification process.

A reading higher than 5 mAbs at 320 nm is indicating that either salt residues or undissolved particles are present in the sample.

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Protein UV Quantification with NanoPhotometer® NP80

Let’s cover the performance of the NanoPhotometer® NP80, top Protein UV Spectrophotometer, in terms of linearity and accuracy, applying small volume protein quantification at 280 nm (Protein UV280). Protein samples display a characteristic absorption spectrum at 280 nm, predominantly from the aromatic amino acids phenylalanine, tyrosine, and tryptophan. The Lambert-Beer law can be applied to determine the protein concentration in a sample. This UV based approach depends strongly on the purity and primary sequence of a protein. Some components which are commonly present in protein samples show strong UV absorbance, and should be corrected for or avoided altogether. The NanoPhotometer® unique feature Blank Control™ will automatically warn the user if high background is present in a blank from buffers or contaminants.

Material & Methods

Bovine serum albumin (BSA) solution A7284 (Lot. SLBP4169V) from Sigma-Aldrich was used as stock solution. Different sample concentrations were achieved by dilution with defined amount of 1x PBS buffer. The dilution ratio was controlled by weight via microbalance (Sartorius BP221S). Expected absorbance values were measured in 10 mm quartz glass cuvettes (Hellma Analytics 100-QS) with UV/VIS spectrometer UVIKON XL (serial number 110178). All measurements were done on the Protein UV Spectrophotometer, NanoPhotometer® NP80 (serial number M80706).

Each protein concentration was measured using Protein UV Spectrophotometer NP80 five times without prior dilution using a sample volume of 1 μl. Each sample was vortexed before every measurement to ensure sample homogeneity. After each measurement, the pedestal and the lid of Protein UV Spectrophotometer NP80 were cleaned with a slightly wet lint free tissue and a new aliquot of the sample was pipetted.

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Precision and Accuracy of the Protein UV Spectrophotometer Measurement Results

Accuracy Results

The expected protein concentration was compared to the mean value of the five measured concentrations of each protein samples (Table 1). Additionally, the standard deviation of the mean absorbance at the 0.67 mm / 0.07 mm path of each sample is listed.

Linearity Results

The resulting linearity curve in the range of 0.0380 – 276.98 mg/ml shows a close correlation between expected and measured concentrations with a coefficient of determination (R2) of 0.9997 (Figure 1). Figure 2 shows the linearity for low concentration samples measured with path length 0.67 (dilution factor 15).

Ratio

The Protein UV Spectrophotometer, NanoPhotometer® NP80,  calculates the 260/280 ratio which accounts for contaminants in the sample. The 260/280 ratio primarily indicates the presence of nucleic acids in the protein sample. Purified protein preparations have an expected ratio of around 0.57. Protein samples used within this technical note had 260/280 ratios between 0.585 and 0.636 for concentrations in a range of 0.705 – 276.8 mg/ml. Figure 3 shows the linearity of high concentrated BSA samples in the range of 68.326 – 276.98 mg/ml.

Table 1. NP80 Expected vs. Measured Protein Concentrations

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Figure 1:
Protein samples (mean value) in the range of
0.038 – 276.98 mg/ml
measured with the NP80 using
automatic path length selection.

Figure 2:
Protein samples (mean value) in the range of
0.038 – 39.097 mg/ml
measured with the NP80 using
path length 0.67 mm.

implen-nanophotometer-protein-UV-measurement-applications-nanodrop-alternative3-linearity-results

Figure 3:
Protein samples (mean value) in the range of
68.326 – 276.98 mg/ml
measured with NP80 using
path length 0.07 mm.

Carryover

Highly concentrated protein samples are known to be sticky. To show that cleaning with just a slightly wet lint-free tissue is appropriate, the blank solution (1x PBS) was measured five times after the final protein sample reading. No residual concentration of protein or absorbance at 280 nm was detected (Figure 4). The absorbance at 280 nm (0.67 mm path) is within the specified fluctuation of the lamp (± 0.002 A).

Figure 4:
No carryover was detected. Absorbance at 280 nm (0.67 mm path) of five 1x PBS measurements measured after the last protein sample

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CHAMPS implen nanophotometer protein UV applications nanodrop alternative CHAPS
T-PER implen nanophotometer protein UV applications nanodrop alternative T-PER
PROTEASE INHIBITORS implen nanophotometer protein UV applications nanodrop alternative PROTEASE INHIBITORS
M-PER implen nanophotometer protein UV applications nanodrop alternative M-PER
NDSB implen nanophotometer protein UV applications nanodrop alternative NDSB
DTT implen nanophotometer protein UV applications nanodrop alternative DTT

RIPA

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HEPES implen nanophotometer protein UV applications nanodrop alternative HEPES

Accuracy and Linearity of the NanoPhotometer®, Protein UV Spectrophotometer

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The presented data illustrate the high accuracy and linearity of the NanoPhotometer® across the entire dynamic range of the instrument. With this novel approach of only two precise path lengths with fixed anchor points utilizing the proprietary True Path Technology™ in a sealed optical environment, mechanical changes in the system are eliminated.

For further technical information please refer also to Technical Note #1 True Path Technology™.

The NanoPhotometer® is the only Protein UV Spectrophotometer with True Path Technology™ providing accurate results without the need for recalibration throughout the entire lifetime of the instrument.

NanoPhotometer® Models that Work Best for Protein UV Quantification

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