细胞生物化学实验教材Exp 1-Spectrophotometry and protein concentration determination-Haibin Zhang.pptxVIP

Lab practice: Cellular biochemistry course Course code：140581 Hours:51 Credits:1.5;Format of lab report;Haibin Tian Division of biochemistry and molecular biology Department of Regenerative medicine Tongji University School of medicine Email: tianhb@tongji.edu.cn ;Overview;【Objectives】 To learn the principle of spectrophotometry. To use spectrophotometry to measure substance concentration in solution. To measure the protein concentration by different method, such as Biuret, BCA, Bradford Assay .;2. Electromagnetic Spectrum;3. Colors and Wavelengths;4.The relationship of substance color and absorption;5. The relationship between absorption or color with substance concentration;(1) Transmittance and Absorbance;A(absorbance) is defined as the negative logarithm of the transmittance.;(2) Lambert – Beer’s law; ;Absorbance A = Log10 = KCL Where: A = amount of light absorbed by the solution expressed as absorbance or optical density Io = intensity of incident light I = intensity of transmitted light K = constant (extinction coefficient ) C = concentration of the substance L = path length of the light-absorbing sample;7. The relationship between A, T and C;8. Methods for analysis of sample concentration;(2) Standard curve method;Optimum wavelength;(2) Choice of Absorbance;(3) Blank reference solution (blank) Since transmittance is a relative measurement, the light transmitted by the sample should be compared to the light transmitted by a reference solution (Blank). The reference solution is generally the solvent in which the colored compound you are interested in is dissolved. A reference is necessary because the solvent itself might absorb some light at the wavelength you are using and you must correct for that absorbance. We assume that the blank transmits 100% of the light entering it that is the scale is set to read zero absorba