Lab 3C Report
University of Texas at Tyler
Lab 3C: Purification of L-Lactate Dehydrogenase
By Affinity Chromatography on Cibacron-Blue Sepharose
David Alexander
10-15-2014
Dr. Black
Chem 4135.001 Abstract: Like the previous experiments, the ultimate goal of this lab was to purify the enzyme sample. However, this is the last lab for purification and high level techniques of purification were employed to achieve this. Dialysis was used first, lowering the small-molecule concentration within the sample. Finally Affinity Chromatography on a Cibacron blue Sepharose stationary phase. Using BSA, which is analogous for BCA assays, a standardization was created to understand where the protein concentration was for each fraction.
Introduction:
This experiment is a continuation of the lab’s efforts to purify L-LDH. Previously our enzyme was purified through anion-exchange chromatography on Q-Sepharose, but to get the enzyme out of the stationary phase NaCl was added to salt it out. However salting out the enzyme prevents using chromatography because of NaCl’s high ionic strength. Dialysis was used to remove the salt from the enzyme sample, when put into a membrane bag which uses principles of equilibrium to remove the salt via nanometer sized holes in the membrane bag which allow for water and small molecule transport. However, dialysis must be done twice, as not all the molecules of salt will leave the dialysis bag because dialysis uses Le Chatelier’s principle equilibrating the bag. The top of the bag has an air pocket introduced to allow for floatation which prevents the stir bar from damaging its contents. For this experiment, affinity chromatography was the main technique used to completely purify L-LDH. Affinity chromatography is exceptional at purification because it uses biospecificity to purify the specific protein. This is achieved by having the stationary phase contain some ligand, substrate-analog, or coenzyme analog of the desired enzyme. For this experiment Cibacron
Lab 3C: Purification of L-Lactate Dehydrogenase
By Affinity Chromatography on Cibacron-Blue Sepharose
David Alexander
10-15-2014
Dr. Black
Chem 4135.001 Abstract: Like the previous experiments, the ultimate goal of this lab was to purify the enzyme sample. However, this is the last lab for purification and high level techniques of purification were employed to achieve this. Dialysis was used first, lowering the small-molecule concentration within the sample. Finally Affinity Chromatography on a Cibacron blue Sepharose stationary phase. Using BSA, which is analogous for BCA assays, a standardization was created to understand where the protein concentration was for each fraction.
Introduction:
This experiment is a continuation of the lab’s efforts to purify L-LDH. Previously our enzyme was purified through anion-exchange chromatography on Q-Sepharose, but to get the enzyme out of the stationary phase NaCl was added to salt it out. However salting out the enzyme prevents using chromatography because of NaCl’s high ionic strength. Dialysis was used to remove the salt from the enzyme sample, when put into a membrane bag which uses principles of equilibrium to remove the salt via nanometer sized holes in the membrane bag which allow for water and small molecule transport. However, dialysis must be done twice, as not all the molecules of salt will leave the dialysis bag because dialysis uses Le Chatelier’s principle equilibrating the bag. The top of the bag has an air pocket introduced to allow for floatation which prevents the stir bar from damaging its contents. For this experiment, affinity chromatography was the main technique used to completely purify L-LDH. Affinity chromatography is exceptional at purification because it uses biospecificity to purify the specific protein. This is achieved by having the stationary phase contain some ligand, substrate-analog, or coenzyme analog of the desired enzyme. For this experiment Cibacron