Protein Purification III Parts
09/15/2015, 09/22/2015, 09/29/2015
Kaisha Butz
Lab Partner: Ryan Van
Abstract
The lab was comprised of three parts, the purpose of which was to separate a protein mix and analyze the results. The first part was separating the proteins; the second part was reacting the proteins with an enzyme, and the third part was analyzing each fraction from Part I to see if separation was successful. It was hypothesized that out of the four proteins in the mix that the glucose oxidase which had the greatest negative charge out of the four proteins would elute last. The hypothesis was supported by the results.
Introduction
Part I: The first lab experiment used ion-exchange chromatography to purify a protein mix. Ion-exchange chromatography is the process of separating charged molecules based on their attraction to the charge of the beads in the column. The experiment used DEAE beads, diethylaminoethyl, which are used for anion exchange. Basically the beads are positive. Once the protein mix is added to the chromatography column the negative ions (anions) will stick to the DEAE beads and the rest will be eluted. The first to elute from the column will be the positive ions (cations) because they will be repelled …show more content…
It was hypothesized that glucose oxidase would elute last from the chromatography column and according to the results from Part III that appears to be the case. Glucose oxidase is made up of two subunits each having a molecular weight of about 80 kilo-Daltons. The third fraction, which contained the last elution material, had a band on the SDS-PAGE gel at around 72 kilo-Daltons. Although alkaline phosphatase has a similar molecular weight it also consists of two subunits making each subunit only 40 kilo-Daltons. Unfortunately the gel bands for wells 8 and 9 were not clear and therefore unable to be
09/15/2015, 09/22/2015, 09/29/2015
Kaisha Butz
Lab Partner: Ryan Van
Abstract
The lab was comprised of three parts, the purpose of which was to separate a protein mix and analyze the results. The first part was separating the proteins; the second part was reacting the proteins with an enzyme, and the third part was analyzing each fraction from Part I to see if separation was successful. It was hypothesized that out of the four proteins in the mix that the glucose oxidase which had the greatest negative charge out of the four proteins would elute last. The hypothesis was supported by the results.
Introduction
Part I: The first lab experiment used ion-exchange chromatography to purify a protein mix. Ion-exchange chromatography is the process of separating charged molecules based on their attraction to the charge of the beads in the column. The experiment used DEAE beads, diethylaminoethyl, which are used for anion exchange. Basically the beads are positive. Once the protein mix is added to the chromatography column the negative ions (anions) will stick to the DEAE beads and the rest will be eluted. The first to elute from the column will be the positive ions (cations) because they will be repelled …show more content…
It was hypothesized that glucose oxidase would elute last from the chromatography column and according to the results from Part III that appears to be the case. Glucose oxidase is made up of two subunits each having a molecular weight of about 80 kilo-Daltons. The third fraction, which contained the last elution material, had a band on the SDS-PAGE gel at around 72 kilo-Daltons. Although alkaline phosphatase has a similar molecular weight it also consists of two subunits making each subunit only 40 kilo-Daltons. Unfortunately the gel bands for wells 8 and 9 were not clear and therefore unable to be