Sweta Soni
2/22/15
Biochem Lab
Experiment 5
Introduction
The purpose of Experiment 5-1 is to separate a mixture of three molecules—blue dextran, cytochrome c, and potassium chromate—through the use of gel-filtration chromatography. The volume at which each molecule elutes will be recorded in order to determine the accuracy of separation. The purpose of Experiment 5-2 is to purify the E. coli enzyme, β-Galactosidase through the use of affinity chromatography on P-aminobenzyl-1-thio-β-D-galactopyranoside agarose, which is a good substrate for the enzyme. This enzyme will be eluted at high pH. The Bradford assay will be used to determine the protein concentration and the specific activity of the solutions containing the enzyme. Size-exclusion chromatography separates molecules …show more content…
For the affinity chromatography, purification of proteins or nucleic acids can be obtained. Affinity chromatography is based on specificity and selectivity because the stationary phase includes an immobilized molecule that can bind to specific proteins. However, this technique results in a highly purified material unlike the size-exclusion technique. Also, affinity chromatography is useful in understanding the enzymatic activity without knowing other physical and chemical properties of the unknown protein. Size-exclusion chromatography is used in the last stages of protein purification while affinity chromatography is used as the first step in purifying proteins. The limitation to size exclusion chromatography is that the sample can be difficult to concentrate due to broadening of the bands of the eluting molecules through the column. While both techniques of chromatography achieve in separating the components within a mixture, the size exclusion chromatography helps determine the molecular mass of the separated compounds and the affinity chromatography is helpful in yielding the highest concentration of protein purification due to its
2/22/15
Biochem Lab
Experiment 5
Introduction
The purpose of Experiment 5-1 is to separate a mixture of three molecules—blue dextran, cytochrome c, and potassium chromate—through the use of gel-filtration chromatography. The volume at which each molecule elutes will be recorded in order to determine the accuracy of separation. The purpose of Experiment 5-2 is to purify the E. coli enzyme, β-Galactosidase through the use of affinity chromatography on P-aminobenzyl-1-thio-β-D-galactopyranoside agarose, which is a good substrate for the enzyme. This enzyme will be eluted at high pH. The Bradford assay will be used to determine the protein concentration and the specific activity of the solutions containing the enzyme. Size-exclusion chromatography separates molecules …show more content…
For the affinity chromatography, purification of proteins or nucleic acids can be obtained. Affinity chromatography is based on specificity and selectivity because the stationary phase includes an immobilized molecule that can bind to specific proteins. However, this technique results in a highly purified material unlike the size-exclusion technique. Also, affinity chromatography is useful in understanding the enzymatic activity without knowing other physical and chemical properties of the unknown protein. Size-exclusion chromatography is used in the last stages of protein purification while affinity chromatography is used as the first step in purifying proteins. The limitation to size exclusion chromatography is that the sample can be difficult to concentrate due to broadening of the bands of the eluting molecules through the column. While both techniques of chromatography achieve in separating the components within a mixture, the size exclusion chromatography helps determine the molecular mass of the separated compounds and the affinity chromatography is helpful in yielding the highest concentration of protein purification due to its