Colorimetry of Micro Spectrophotometer
BCA, Lowry and Bradford all use colorimetric methods to detect the concentration of impurity protein. A standard curve must be constructed when the colorimetric method is used to detect the protein concentration, so these three methods of measuring protein are combined into the colorimetric method.
BCA is a colorimetric method for detecting the concentration of impure proteins. It is commonly used to detect the concentration of diluted proteins and those containing impurities that absorb light in the ultraviolet region. BCA method is a method to detect Cu+1 ions. Under alkaline environment, Cu+2 ions will be reduced to Cu+1 ions by proteins. Two BCA molecules of biquinoline dicarboxylate and a Cu+1 ion form the purple chelate such that in the presence of the protein, the chelate formed by Cu-BCA has the maximum light absorption at 562nm, normalized to the light coefficient of 750nm.
Commercial BCA kits have two protein detection ranges:
Conventional tests use a reagent/protein sample volume ratio of 20:1. The kit ranges from 0.20mg/ mL to 8.0mg/ mL (BSA). When using a base test, it is recommended to use 4ul samples and 80ULBCA reagents.
Minimal detection using a 1:1 reagent/sample can detect protein concentrations ranging from 0.01mg/ mL to 0.20mg/m. To prepare enough samples for the base test, it is recommended to use a 10ul sample and a 10ULBCA reagent (using a P tube).
Establish standard curves and sample preparation as recommended by the kit manufacturer. Ensure that the same time and temperature are used for all tests.
Note: if the test temperature is higher than 60 degrees, it is best to use twice the test volume to avoid volatilization resulting in sample reduction, which will affect the test results.
Lowry method is a widely used method for protein quantification. Lowry method is the reaction of protein with copper sulfate in an alkaline environment to form copper-protein complex. The Folin-Ciocalteu reagent effectively reduces the copper complex, producing a blue product that is proportional to the amount of protein and can be detected at 650nm and calibrated at 405nm. The reagents used in the tests can be purchased from multiple manufacturers.
For accurate preparation of the standard, it is recommended to use 20ul protein samples and 100ul Lowry reagent for the reaction. The sample concentration range that can be detected on this instrument is from 0.20mg/ mL to 4mg/ mL. Prepare standard and sample according to kit manufacturer's instructions. The treatment time of each sample should be consistent with the ambient temperature during all operations. Since the concentration range detected by the base of the instrument is larger than that of conventional tests, it is necessary to construct a wider range of standard curves than recommended by the manufacturer when constructing standard curves. 2ul is recommended for the sample quantity required for testing.
Bradford is a commonly used method of protein quantification. It is usually used to detect the concentration of proteins in low concentrations. Bradford method detects the protein concentration according to the protein's ability to make Coomassian Bright Blue attempt to absorb the shift, and generally detects the absorbance at 595nm. The protein-dye complex was detected at 595nm and standardized at 750nm. Corresponding kits can be purchased from multiple manufacturers.
Commercial Bradford kits have two protein detection ranges:
Conventional tests use a 50:1 reagent/protein sample volume ratio, and this kit ranges from 0.10mg/ mL to 8.0mg/ mL (BSA). The optimal linear range was 0.01-1mg/ mL. When using a base test, it is recommended to use 4ul of the sample and 200ul of Bradford reagent.
Microassay uses a 1:1 reagent/sample to detect protein concentrations ranging from 15ug/ml to 125ug/ml. To prepare enough samples for the base test, it is recommended to use 10ul samples and 10ULBCA reagent (using PCR tube). Establish standard curves and sample preparation as recommended by the kit manufacturer. Ensure that the same time and temperature are used for all tests.
Note: if the test temperature is higher than 60 degrees, it is best to use twice the test volume to avoid volatilization resulting in sample reduction, which will affect the test results.
The Bradford kit also includes a standard substance used to construct a standard curve. Because the instrument can detect higher concentrations than conventional dish detection, users need to use a higher concentration than the manufacturer's recommended standard.
Colorimetric detection
Note: the colorimetric test before the sample test, need to establish a standard curve!
