2009_Lab__1_-_Spec._Quantification.ppt

# 2009_Lab__1_-_Spec._Quantification.ppt - Quiz Lab 1 Spec....

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Quiz Lab 1 Spec…. . Your name The Bradford assay is used to measure the concentration In the expression of the Beer-Lambert law, The natural substrate in bacterial cells of the enzyme of __________ in solution. (1 pt) A = ε l c what do the three terms on the right stand for? (3 pts) beta-galactosidase is ___________ . (1 pt)

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Objectives: To develop a familiarity with spectrophotometry and operations of spectrophotometer. - Beer-Lambert Law - Operate the spectrophotometer To construct two standard curves (using linear regression model) - The importance of a standard curve - Standard curve for o-NP - Standard curve for Bradford protein assay Spectrophotometric Quantification
Principle of Spectrophotometer 1. White light separated by prism to different wavelengths. 2. Wavelength at which absorbance is at a maximum is selected. 3. Transmitted light is collected by a photoelectric tube. 4. Transmitted light energy is converted to electricity. 5. A galvanometer reads the electricity.

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%T = light intensity transmitted by sample (I 1 ) x 100 light intensity transmitted by blank (I o ) %T is NOT directly proportional to the concentration of absorbing substances. Absorbance is directly proportional to absorbing substances. The relationship between absorbance (A) and %T is given by: A = log(100/%T) or A = 2 – log(%T) Relationship between %T and Absorbance (A)
The concentration of a solution is determined using the relationship known as Beer’s Law : A = ε l c A: absorbance (no unit) : extinction coefficient (L/mole*cm) l: path length (1 cm) c: concentration (mole/L) This relationship states that the absorbance of light at a

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## This note was uploaded on 03/11/2010 for the course BISC 330L taught by Professor Petruska,tower during the Spring '07 term at USC.

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2009_Lab__1_-_Spec._Quantification.ppt - Quiz Lab 1 Spec....

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