Micro SpectrophotometeFunctional advantages of.
Micro Spectrophotometer not only covers the functions of visible light Spectrophotometer, but also can carry out the application of ultraviolet Spectrophotometer. The most commonly used aspects are as follows:
Quantification of nucleic acid
Nucleic acid quantification is the most frequently used function of the Micro Spectrophotometer. The buffer-soluble oligonucleotides, single-stranded DNA, double-stranded DNA, and RNA can be quantified. The absorption wavelength of the highest absorption peak of nucleic acid is 260nm. Each nucleic acid has a different molecular composition, so its conversion coefficient is different. To quantify different types of nucleic acids, the corresponding extinction factor should be selected in advance. For example, the absorption value of 1OD is equivalent to 50μg/ mL of dsDNA, 37μg/ mL of ssDNA, 40μg/ mL of RNA, 30μg/ mL of OLIG. The absorption value after the test is converted by the above coefficients to obtain the corresponding sample concentration. In addition to the nucleic acid concentration, the spectrophotometer showed several very important ratios indicating the purity of the sample, such as the A260/A280 ratio, which is used to assess the purity of the sample since the protein absorption peak is 280nm. For pure samples, the ratio is greater than 1.8(DNA) or 2.0(RNA). A ratio below 1.8 or 2.0 indicates a protein or phenolic influence. A230 indicates that there are some pollutants in the sample, such as carbohydrates, polypeptides, phenols, etc. The A260/A230 ratio of the relatively pure nucleic acid is greater than 2.0.
UV-Vis routine visible - ultraviolet detection
The full wavelength Micro Spectrophotometer can function just like a normal UV-Vis Spectrophotometer. For example, the Bio-DL MicroDrop can display the absorbance value of the sample at light waves ranging from 185 to 910nm. Up to 40 wavelengths can be detected at the same time and the absorption values can be specified and displayed in the report.
Direct quantification of protein (UV method)
This method directly tests the protein at 280nm. The protein determination process is very simple, first test the blank fluid, then test the protein directly. The direct protein quantification method is suitable for the determination of relatively pure protein with relatively single composition. Compared with colorimetric method, UV direct quantitative method is fast and easy to operate. But susceptible to interference by parallel substances, such as DNA; In addition, the sensitivity is low and the protein concentration is high.
Protein quantification by colorimetric method
Protein is usually a compound of multiple proteins. The basis of colorimetry is the composition of protein: 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 in the protein reaction, thus reflecting the protein concentration.
Colorimetric methods generally have BCA, Bradford, Lowry and other methods.
Lowry method: Based on and improved upon the earliest Biuret reaction. Proteins react with Cu2+ to produce blue reactants. However, Lowry's method is more sensitive than Biuret's. The disadvantage is that several different reaction reagents need to be added in sequence; The reaction time is longer; Susceptible to non-protein substances; The proteins containing EDTA, TritonX-100, Ammoniasulfate and other substances are not suitable for this method.
BCA(Bicinchonine Acidassay) method: This is a new and more sensitive protein assay. 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 562nm. The linear relationship between this compound and protein concentration is very strong, and the compound formed after the reaction is very stable. Compared with Lowry method, the operation is simple and sensitive. But similar to Lowry's method