UV-1200 spectrophotometer in the measurement of nucleic acid protein - Database & Sql Blog Articles

Application of spectrophotometer in nucleic acid protein measurement

Spectrophotometers have become routine instruments in modern molecular biology laboratories. Often used for nucleic acid, protein quantification and quantification of bacterial growth concentrations.
The simple principle of the spectrophotometer The spectrophotometer uses a light source that can generate multiple wavelengths, and through a series of spectroscopic devices, to generate a specific wavelength of light source, after the light source passes through the tested sample, part of the light source is absorbed, and the absorbance of the sample is calculated. Thereby converting to the concentration of the sample. The absorbance of the sample is proportional to the concentration of the sample.

The quantification of nucleic acid quantitative nucleic acids is the most frequently used function of the spectrophotometer. Oligonucleotides, single-stranded, double-stranded DNA, and RNA can be quantified in buffer. The absorption peak of the highest absorption peak of nucleic acid is 260 nm. The molecular composition of each nucleic acid is different, so the conversion factor is different. To quantify different types of nucleic acids, select the corresponding coefficients in advance. For example, the absorbance of 1 OD corresponds to 50 μg/ml of dsDNA, 37 μg/ml of ssDNA, 40 μg/ml of RNA, and 30 μg/ml of Olig. The absorbance after the test is converted by the above coefficients to obtain the corresponding sample concentration. Before testing, select the correct procedure, enter the volume of the stock solution and diluent, and then test the blank and sample solution. However, the experiment was not always smooth. Unstable readings may be the biggest headache for the experimenter. Instruments with higher sensitivity show greater drift in absorbance.

In fact, the design principle and working principle of the spectrophotometer allow the absorbance to vary within a certain range, that is, the instrument has certain accuracy and precision. For example, the accuracy of the Eppendorf Biophotometer is ≤1.0% (1A). The results of such multiple tests vary between a mean of 1.0% and are normal. In addition, it is also necessary to consider the physicochemical properties of the nucleic acid itself and the pH of the buffer in which the nucleic acid is dissolved, the ion concentration, etc.: When the ion concentration is too high during the test, the reading shifts, so it is recommended to use a certain pH value and a low ion concentration. Buffers, such as TE, 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, especially nucleic acid samples, in the sample. The presence of these small particles interferes with the test results. In order to minimize the effect of the particles on the test results, the absorbance of the nucleic acid is required to be at least greater than 0.1 A, and the absorbance is preferably between 0.1 and 1.5 A. Within this range, the interference of the particles is relatively small and the results are stable.

This means that the concentration of the sample should not be too low or too high (beyond the test range of the photometer). Finally, the operating factors, such as mixing should be sufficient, otherwise the absorbance value is too low, even negative values; the mixture can not exist bubbles, the blank liquid has no suspended matter, otherwise the reading drifts sharply; the same cuvette must be used to test the blank and sample Otherwise, the difference in concentration is too large; the conversion factor and sample concentration unit are selected consistently; the cuvette with window wear cannot be used; the volume of the sample must meet the minimum volume required for the cuvette.

In addition to the nucleic acid concentration, the spectrophotometer also shows several very important ratios indicating the purity of the sample, such as the ratio of A 260 / A 280, used to assess the purity of the sample because the absorption peak of the protein is 280 nm. A pure sample with a ratio 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. A 230 indicates that some contaminants are present in the sample, such as carbohydrates, polypeptides, phenols, etc., and the ratio of the purer nucleic acid A 260 / A 230 is greater than 2.0. A 320 detects the turbidity of the solution and other interference factors. For pure samples, A 320 is generally 0.

Direct quantification of proteins (UV method)
This method is a direct test of 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 appropriate conversion method to convert the absorbance to the sample concentration.

The protein determination process is very simple, first test the blank and then test the protein directly. Due to some impurities in the buffer, it is common to eliminate the "background" information of 320 nm and set this function to "on". Similar to the test nucleic acid, the absorbance of A 280 is required to be at least greater than 0.1 A, and the optimum linear range is between 1.0 and 1.5. When the Warburg formula was selected in the experiment to show the sample concentration, the reading was found to "drift". This is a normal phenomenon. In fact, as long as the change in the absorbance of A 280 does not exceed 1%, the results are very stable.
The reason for the drift is because the absorbance value of the Warburg formula is converted into a concentration, multiplied by a certain coefficient, as long as the absorbance value is slightly changed, the concentration is amplified, and the result is unstable.

A direct protein quantification method for testing relatively pure, relatively single-component proteins. Compared with the colorimetric method, the ultraviolet direct quantitative method is fast and easy to operate; but it is easily interfered by parallel substances, such as DNA interference; in addition, the sensitivity is low, and the protein concentration is required to be high. Colorimetric Proteins Proteins are usually compounds of a variety of proteins. Colorimetric assays are based on protein constituents: amino acids (such as tyrosine, serine) react with additional chromogenic groups or dyes to produce colored materials. The concentration of the colored substance is directly related to the number of amino acids reacted by the protein, thereby reacting 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. However, the Lowry method is more sensitive than Biuret. The disadvantage is that several different reagents need to be added sequentially; the reaction takes a long time; it is susceptible to non-protein substances; proteins containing substances such as EDTA, Triton x-100, ammonia sulfate are not suitable for this method. BCA (Bicinchoninine acid assay): This is a newer, more sensitive protein test. 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 562 nm. This compound has a strong linear relationship with protein concentration, and the compound formed after the reaction is very stable. Compared with the Lowry method, the operation is simple and the sensitivity is high. However, 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 of 595 nm. Its biggest feature is that it has good sensitivity, which is twice as high as Lowry and BCA; it is simpler and faster; only one reagent is needed; the compound can be stable for 1 hour, which is convenient for the result; The reducing agent (such as DTT, mercaptoethanol) which interferes with Lowry, BCA reaction is compatible. But it is still sensitive to detergents. The main disadvantage is that different standards can lead to large differences in the results of the same sample, which is incomparable. Some researchers who have first-time colorimetric assays may be inconsistent with the results of various colorimetric methods, and are confused. Which method should I believe? Since the groups reacted by the various methods and the chromogenic groups are different, the concentration of the sample obtained by the same sample is incomparable at the same time using several methods. For example, Keller et al. tested the protein in human milk. As a result, the concentration measured by Lowry and BCA was significantly higher than that of Bradford, and the difference was significant. Even if the same sample was measured, the standard samples selected by the same colorimetric method were inconsistent, and the concentrations after the test were inconsistent. If the protein in the cell homogenate is tested with Lowry, BSA is used as a standard at a concentration of 1.34 mg / ml, and a globulin is used as a standard at a concentration of 2.64 mg / ml. Therefore, before selecting the colorimetric method, it is preferable to refer to the chemical composition of the sample to be tested, and to find a standard protein having a similar chemical composition as a standard. In addition, colorimetric methods for quantifying proteins often have problems in that the absorbance of the sample is too low, resulting in a large difference between the measured sample concentration and the actual concentration. The key issue 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 the standard sample) must be tested during this time. When the time is too long, the obtained absorbance value becomes small, and the converted concentration value decreases. In addition, the reaction temperature, pH value of the solution, etc. are all important reasons for the experiment. In addition, it is very important to use plastic colorimetry. Avoid using quartz or glass cuvettes because the color of the reaction will stain the quartz or glass, resulting in inaccurate sample absorbance. Bacterial Cell Density (OD 600) The laboratory determines the bacterial growth density and growth phase, and infers the growth density of the bacteria based on experience and visual observation. In the case of more demanding experiments, it is necessary to accurately determine the bacterial cell density using a spectrophotometer. OD600 is the standard method for tracking microbial growth in liquid cultures. The culture solution containing no bacterial solution was used as a blank solution, and then the culture-containing culture solution after the culture was quantitatively determined. In order to ensure proper operation, the cell count must be performed with a microscope for each microorganism and each instrument to make a calibration curve. Occasionally, there is a negative value of the OD value of the bacterial liquid in the experiment, because the color developing medium is used, that is, after the bacteria are cultured for a while, it reacts with the medium to cause a color change reaction. In addition, it should be noted that the sample tested cannot be centrifuged to maintain the bacterial suspension.