By David W. Burden, Donald B. Whitney (auth.)
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Extra info for BiotechnologyProteins to PCR : A Course in Strategies and Lab Techniques
Carlsbergensis will be assessed in order to determine the most appropriate medium for the production of the enzyme. , hydrolysis of galactose-containing sugars such as melibiose and tachyose) will be measured by hydrolysis of the substrate, p-nitrophenyl-a-D-galactoside. Additionally, the proteins present in the yeast broth will be measured by several colorimetric based assays to determine total protein concentration. 2 BACKGROUND Proteins are large, polymeric biomolecules that are composed of a amino acids covalently linked through peptide bonds.
Proteins tend to stick to most surfaces, and this can be a serious source of protein loss during manipulation and handling, especially with dilute solutions. Loss of protein in this way can be minimized by maintaining a high concentration or by adding a second inert protein, such as albumin, which increases the overall protein concentration. The inert protein should be one that can be easily removed later on. Glassware or plasticware used in protein studies should be meticulously washed and rinsed with deionized water.
A variation of Beer's Law is used to determine protein concentration in mg/ml. 2. 2 Absorbance =(absorption constant) ml mg-1em-1 x Concentration x 1 em therefore: Absorbance/absorption constant =concentration (mg/ml) The absorption constant is specific for the particular protein being measured and is determined using a cuvette with a 1 cm path length containing a protein solution of known concentration (in mg/ ml). 3 is used. , 1 OD = 1 mg/ml protein). This measurement is considered qualitative since a true molar extinction coefficient or absorption constant is not used.
BiotechnologyProteins to PCR : A Course in Strategies and Lab Techniques by David W. Burden, Donald B. Whitney (auth.)