Introduction
Affinity Chromatography, also referred to as biospecific chromatography, is a technique of partitioning rooted in biological affinity (Dean). This practice capitalizes on complex interactions such as bond formation between enzymes and substrate, or more broadly: macromolecules and ligands. Purification is at the center of the use of affinity chromatography. In general, samples may be run through a gel that contains a corresponding component that binds with the sample at certain conditions. These conditions vary from acidity level, contending ligands, to ionic strength (Dean). Following the formation of the desired interaction, another component (a buffer) is used to wash unwanted elements that may also be in the column. The column …show more content…
Trypsin is also known to act on itself resulting in both active and inactive forms of trypsin during storage (Sugumaran). In this experiment, affinity chromatography was used to extrapolate the active form of trypsin from the inactive form of trypsin. A column containing P-amino-bezaidine was used to form a bond with the active form of trypsin in the matrix (Sugumaran). Two assays were performed. The first assay was to determine if there was any active trypsin by employing Benyzol-DL-arginie-p-nitroanilide (BAPNA), which forms an observable yellow color (from p-nitroaniline) when cleaved by active trypsin. The second assay used biorad protein assay reagent to estimate the concentration of the protein that was eluted. Both assays required a spectrometer to observe the color changes by absorbance. The hypothesis is that active and inactive tryspin would be able to be separated by affinity chromatography according to knowledge about binding and the regents …show more content…
Figure 3, Affinity Column: Protein Concentration and Enzyme Activity, shows the two peaks for protein concentrations on the red line. The blue line, showing the enzymes activity, overlaps with test tube six that contained the active trypsin. The lack of overlap over test tube one combined with a peak for a protein concentration identifies the inactive trypsin. Using the equation in the sample calculation of trypsin percentage, it was found that there was 75.6% yield of trypsin.
Future labs could benefit from having consistent samples of trypsin and Biorad. The conditions from the trypsin and biorad must remain the same throughout the experiment. Other suggestions include making dilutions from the original fractions and not from a separate test tube as that introduces new variables to the mix such as an uneven distribution of trypsin when making dilutions in a new tube.
This experiment further dove into the multiple application of affinity chromatography such as examining the biological interactions of antigens, hormones and other elements. One example of this use is by Dr. Chi Zhang to purify antibodies. Antibodies have multiple uses especially in the medical field. Her research analyzed a way to purify a relatively large amount of sample overnight (Zhang). Thought the lab experiment focused on trypsin, the broad use of affinity chromatography should not be
Affinity Chromatography, also referred to as biospecific chromatography, is a technique of partitioning rooted in biological affinity (Dean). This practice capitalizes on complex interactions such as bond formation between enzymes and substrate, or more broadly: macromolecules and ligands. Purification is at the center of the use of affinity chromatography. In general, samples may be run through a gel that contains a corresponding component that binds with the sample at certain conditions. These conditions vary from acidity level, contending ligands, to ionic strength (Dean). Following the formation of the desired interaction, another component (a buffer) is used to wash unwanted elements that may also be in the column. The column …show more content…
Trypsin is also known to act on itself resulting in both active and inactive forms of trypsin during storage (Sugumaran). In this experiment, affinity chromatography was used to extrapolate the active form of trypsin from the inactive form of trypsin. A column containing P-amino-bezaidine was used to form a bond with the active form of trypsin in the matrix (Sugumaran). Two assays were performed. The first assay was to determine if there was any active trypsin by employing Benyzol-DL-arginie-p-nitroanilide (BAPNA), which forms an observable yellow color (from p-nitroaniline) when cleaved by active trypsin. The second assay used biorad protein assay reagent to estimate the concentration of the protein that was eluted. Both assays required a spectrometer to observe the color changes by absorbance. The hypothesis is that active and inactive tryspin would be able to be separated by affinity chromatography according to knowledge about binding and the regents …show more content…
Figure 3, Affinity Column: Protein Concentration and Enzyme Activity, shows the two peaks for protein concentrations on the red line. The blue line, showing the enzymes activity, overlaps with test tube six that contained the active trypsin. The lack of overlap over test tube one combined with a peak for a protein concentration identifies the inactive trypsin. Using the equation in the sample calculation of trypsin percentage, it was found that there was 75.6% yield of trypsin.
Future labs could benefit from having consistent samples of trypsin and Biorad. The conditions from the trypsin and biorad must remain the same throughout the experiment. Other suggestions include making dilutions from the original fractions and not from a separate test tube as that introduces new variables to the mix such as an uneven distribution of trypsin when making dilutions in a new tube.
This experiment further dove into the multiple application of affinity chromatography such as examining the biological interactions of antigens, hormones and other elements. One example of this use is by Dr. Chi Zhang to purify antibodies. Antibodies have multiple uses especially in the medical field. Her research analyzed a way to purify a relatively large amount of sample overnight (Zhang). Thought the lab experiment focused on trypsin, the broad use of affinity chromatography should not be