Abstract: Spectrophotometer has become a routine instrument in modern molecular biology laboratory. Commonly used for quantification of nucleic acid, protein and bacterial growth concentration.

Simple principle of spectrophotometer

The spectrophotometer uses a light source that can generate multiple wavelengths. A series of spectroscopic devices are used to generate a light source of a specific wavelength. After the light source passes through the tested sample, part of the light source is absorbed, and the absorbance value of the sample is calculated to be converted into the sample concentration . The absorbance of the sample is proportional to the concentration of the sample.

Quantification of nucleic acids

The quantification of nucleic acid is the most frequently used function of spectrophotometer. It can quantify oligonucleotides, single-stranded and double-stranded DNA, and RNA dissolved in buffer. The absorption wavelength of the highest absorption peak of nucleic acid is 260 nm. Each nucleic acid has a different molecular composition, so its conversion factor is different. To quantify different types of nucleic acids, the corresponding coefficients must be selected in advance. For example: 1OD absorbance value is equivalent to 50μg / ml dsDNA, 37μg / ml ssDNA, 40μg / ml RNA, 30μg / ml Olig. The absorbance value after the test is converted by the above-mentioned coefficients to obtain the corresponding sample concentration. Before testing, select the correct program, enter the volume of the original solution and diluent, and then test the blank solution and sample solution. However, the experiment was not smooth. Unstable readings may be the biggest headache for experimenters. The higher the sensitivity of the instrument, the greater the shift in absorbance.

In fact, the design principle and working principle of the spectrophotometer allow the absorbance value to change within a certain range, that is, the instrument has a certain accuracy and precision. For example, the accuracy of EppendorfBiophotometer is ≤1.0% (1A). It is normal for the results of such multiple tests to vary between about 1.0% average. In addition, the physicochemical properties of the nucleic acid itself and the pH value and ion concentration of the nucleic acid-dissolving buffer should also be considered: during the test, too high ion concentration will also cause the reading to drift. Buffers, such as TE, can greatly stabilize readings. The dilution concentration of the sample is also a factor that cannot be ignored: due to the inevitable presence of some fine particles in the sample, especially nucleic acid samples. The presence of these small particles interferes with the test effect. In order to minimize the impact of the particles on the test results, it is required that the nucleic acid absorbance value is at least greater than 0.1A, and the absorbance value is preferably 0.1-1.5A. Within this range, the interference of the particles is relatively small, and the results are stable.

This means that the concentration of the sample cannot be too low or too high (beyond the test range of the photometer). Finally, there are operational factors, such as sufficient mixing, otherwise the absorbance value is too low, or even a negative value; there can be no bubbles in the mixed liquid, no suspension in the blank liquid, otherwise the reading drifts drastically; the blank and sample must be tested with the same cuvette Otherwise, the concentration difference is too large; the conversion coefficient and the sample concentration unit are selected to be consistent; the cuvette with window wear cannot be used; the volume of the sample must reach the minimum volume required by the cuvette and other operational matters.

In addition to the nucleic acid concentration, the spectrophotometer also displays several very important ratios indicating the purity of the sample, such as the A260 / A280 ratio, which is used to evaluate the purity of the sample because the absorption peak of the protein is 280 nm. For pure samples, the ratio is greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is lower than 1.8 or 2.0, it indicates the presence of protein or phenolic substances. A230 means that there are some contaminants in the sample, such as carbohydrates, peptides, phenol, etc., the ratio of the pure nucleic acid A260 / A230 is greater than 2.0. A320 detects the turbidity of the solution and other interference factors. Pure samples, A320 is generally 0.

Direct quantification of protein (UV method)

This method directly tests the protein at a wavelength of 280 nm. Select the Warburg formula, the photometer can directly display the concentration of the sample, or select the corresponding conversion method to convert the absorbance value to the sample concentration.

The protein determination process is very simple, first test the blank solution, and then directly test the protein. Due to the presence of some impurities in the buffer, it is generally necessary to eliminate the "background" information at 320 nm and set this function to "ON". Similar to the test nucleic acid, the absorbance of A280 is required to be at least greater than 0.1A, and the optimal linear range is between 1.0-1.5. When the Warburg formula was selected to display the sample concentration during the experiment, the reading "drifted" was found. This is a normal phenomenon. In fact, as long as the change range of the absorbance value of A280 is not more than 1%, the result is very stable.

The reason for the drift is that the absorbance value of Warburg formula is converted into concentration and multiplied by a certain coefficient. As long as the absorbance value changes a little, the concentration will be amplified, so the result is very unstable.

The direct protein quantification method is suitable for testing relatively pure proteins with relatively single components. Compared with the colorimetric method, the UV direct quantitative method is faster and easier to operate; but it is susceptible to interference from parallel substances, such as DNA; in addition, the sensitivity is low and the protein concentration is required to be high.

Colorimetric protein quantification

Protein is usually a compound of a variety of proteins. The basis of colorimetric determination is the protein constituents: amino acids (such as tyrosine, serine) react with additional chromogenic groups or dyes to produce colored substances. The concentration of the colored substance is directly related to the number of amino acids that the protein reacts with, thereby reflecting the protein concentration.

Colorimetric methods generally include BCA, Bradford, Lowry and other methods.

Lowry method: based on the earliest Biuret reaction and improved. The protein reacts with Cu2 to produce a blue reactant. But compared with Biuret, the Lowry method is more sensitive. The disadvantage is that several different reagents need to be added in sequence; the reaction takes a long time; it is easily affected by non-protein substances; proteins containing EDTA, Tritonx-100, ammoniasulfate and other substances are not suitable for this method.

BCA (Bicinchoninineacidassay) method: This is a newer and more sensitive protein test method. The protein to be analyzed reacts with Cu2 in an alkaline solution to produce Cu, which forms a chelate with BCA to form a purple compound with an absorption peak at a wavelength of 562nm. The linear relationship between this compound and protein concentration is extremely strong, and the compound formed after the reaction is very stable. Compared with the Lowry method, the operation is simple and the sensitivity is high. But similar to the Lowry method, it is susceptible to interference between proteins and detergents.

Bradford method: The principle of this method is that the protein reacts with Coomassie Brilliant Blue to produce a colored compound with an absorption peak at 595 nm. Its biggest feature is that it has good sensitivity, which is twice that of Lowry and BCA test methods; the operation is simpler and faster; only one reaction reagent is needed; the compound can be stable for 1 hour, which is convenient for results; Reducing agents (such as DTT, mercaptoethanol) that interfere with Lowry and BCA reactions are compatible. But it is still sensitive to detergents. The main disadvantage is that different standards will cause the results of the same sample to be very different and incomparable.

Some researchers who are initially exposed to colorimetric methods may be confused by the results of various colorimetric methods. They are confused, which method should be believed? Because the groups reacted by various methods and the color developing group are different , So using several methods at the same time is incomparable for the sample concentration obtained from the same sample. For example: Keller et al. Tested the protein in human milk and found that the concentration measured by Lowry and BCA was significantly higher than that of Bradford, and the difference was significant. Even if the same sample is measured, the standard sample selected by the same colorimetric method is inconsistent, and the concentration after the test is also inconsistent. For example, use Lowry to test the protein in the cell homogenate, using BSA as the standard, with a concentration of 1.34mg / ml, and using aglobulin as the standard, with a concentration of 2.64mg / ml. Therefore, before choosing a colorimetric method, it is best to refer to the chemical composition of the sample to be tested and look for a standard protein with a similar chemical composition as a standard. In addition, when quantifying protein by colorimetry, the problem often occurs is that the absorbance value of the sample is too low, resulting in a large gap between the measured sample concentration and the actual concentration. The key problem is that the color after the reaction has a certain half-life, so each colorimetric method lists the reaction test time, and all samples (including standard samples) must be tested within this time. If the time is too long, the obtained absorbance value becomes smaller, and the converted concentration value decreases. In addition, the reaction temperature, the pH value of the solution, etc. are all important reasons that affect the experiment. In addition, it is very important to use plastic colorimetry. Avoid using cuvettes made of quartz or glass, because the color of the reaction will make the quartz or glass color, resulting in inaccurate absorbance of the sample.

Bacterial cell density (OD600)

The laboratory determines the bacterial growth density and growth period, and infers the bacterial growth density based on experience and visual observation. When encountering more demanding experiments, it is necessary to use a spectrophotometer to accurately determine the bacterial cell density. OD600 is the standard method for tracking the growth of microorganisms in liquid culture. The culture solution without the bacterial solution was used as a blank solution, and then the bacterial-containing culture solution after the cultivation was quantified. In order to ensure correct operation, a microscope must be used to count cells for each microorganism and each instrument, and a calibration curve must be made. Occasionally, the OD value of the bacterial solution will appear negative in the experiment, because the color-developing medium is used, that is, after the bacteria are cultured for a period of time, the medium reacts with the medium and a color change occurs. In addition, it should be noted that the tested sample cannot be centrifuged to keep the bacteria suspended.

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