Protein Separation Using Gel Filtration Chromatography
Introduction:
Chromatography techniques are one of the most useful methods use for the isolation or separation of macromolecule from a mixture. The most common used chromatography techniques are thin -layer chromatography (TLC), column chromatography, etc. The purpose of this lab exercise is using the gel filtration chromatography to separate different proteins from a mixture based on their size. Also, collecting elutes that run through the column, and constructing an elution profile to show how the column separates molecules by molecular weight. Gel filtration chromatography is one type of column chromatography, which separates molecules according to their size (#1 Lehninger, 87). It …show more content…
Cytochrome C has a size of 13,000 Da, pink in color. Potassium Chromate has a size of 192 Da, yellow in color. According to the theory of Gel Filtration Chromatography, the Blue Dextran will elute first because its size is bigger than the exclusion size of the resin (2x106 Da), and cannot enter the pore of the bead. Therefore, it will travel rapidly and eluted sooner. The Cytochrome will elute from the column next because its size is not bigger than the exclusion size of the resin, but not small enough to enter in many pores in the gel, we can call it a medium sized molecule. Medium sized molecules enter some pores of the bead, so it moves and elute slower than the bigger size molecules. Potassium Chromate will elute out of the column last due to its smallest size (192 Da) compare to other 2 proteins. Very small molecules enter and fit in many pores of the bead. Thus, they are retained and move slower through the column. In addition, there should be 3 peaks when graphing the data that collected. The first peak should be the Blue Dextran, second peak should be Cytochrome C, and the last peak should be the Potassium Chromate according to their different sizes and the order of …show more content…
The ring stand and the clean, dry column were obtained. The column was clamped to the ring vertically and the stopcock at the bottom was turned horizontally with respect to the column. The stopcock turned horizontally that means it is locked prevent the buffer from drain out the column. 4mL of buffer (20 mM sodium phosphate, pH 7) was measured by using the P-1000 micropipette and was added in the column. The G-25 slurry was obtained by the TA, and was carefully poured into the column. After the slurry added, the stopcock was turned vertically with respect to the column in order to open the valve to allow the slurry to settle and solvent to drain until it reaches about 1cm above the bed. Next, equilibrate the packed column by by adding 20mL in total of 20 mM Sodium Phosphate, pH 7 buffer. 5 mL of buffer was measured by the P-1000 and transferred into the column each time to prevent overflowing. The buffer was allowed to run through the column until it reaches about 1cm above the bed. During every part of the experiment, it is very important not to let the bed run dry because the pockets of air in the bed will cause an uneven flow of molecules through the column. The packed column was stored for the second day
Introduction:
Chromatography techniques are one of the most useful methods use for the isolation or separation of macromolecule from a mixture. The most common used chromatography techniques are thin -layer chromatography (TLC), column chromatography, etc. The purpose of this lab exercise is using the gel filtration chromatography to separate different proteins from a mixture based on their size. Also, collecting elutes that run through the column, and constructing an elution profile to show how the column separates molecules by molecular weight. Gel filtration chromatography is one type of column chromatography, which separates molecules according to their size (#1 Lehninger, 87). It …show more content…
Cytochrome C has a size of 13,000 Da, pink in color. Potassium Chromate has a size of 192 Da, yellow in color. According to the theory of Gel Filtration Chromatography, the Blue Dextran will elute first because its size is bigger than the exclusion size of the resin (2x106 Da), and cannot enter the pore of the bead. Therefore, it will travel rapidly and eluted sooner. The Cytochrome will elute from the column next because its size is not bigger than the exclusion size of the resin, but not small enough to enter in many pores in the gel, we can call it a medium sized molecule. Medium sized molecules enter some pores of the bead, so it moves and elute slower than the bigger size molecules. Potassium Chromate will elute out of the column last due to its smallest size (192 Da) compare to other 2 proteins. Very small molecules enter and fit in many pores of the bead. Thus, they are retained and move slower through the column. In addition, there should be 3 peaks when graphing the data that collected. The first peak should be the Blue Dextran, second peak should be Cytochrome C, and the last peak should be the Potassium Chromate according to their different sizes and the order of …show more content…
The ring stand and the clean, dry column were obtained. The column was clamped to the ring vertically and the stopcock at the bottom was turned horizontally with respect to the column. The stopcock turned horizontally that means it is locked prevent the buffer from drain out the column. 4mL of buffer (20 mM sodium phosphate, pH 7) was measured by using the P-1000 micropipette and was added in the column. The G-25 slurry was obtained by the TA, and was carefully poured into the column. After the slurry added, the stopcock was turned vertically with respect to the column in order to open the valve to allow the slurry to settle and solvent to drain until it reaches about 1cm above the bed. Next, equilibrate the packed column by by adding 20mL in total of 20 mM Sodium Phosphate, pH 7 buffer. 5 mL of buffer was measured by the P-1000 and transferred into the column each time to prevent overflowing. The buffer was allowed to run through the column until it reaches about 1cm above the bed. During every part of the experiment, it is very important not to let the bed run dry because the pockets of air in the bed will cause an uneven flow of molecules through the column. The packed column was stored for the second day