Gel Electrophoresis Lab
Ali A. Mohammed
Cell Structure and Function
16 October 2014
Gel Filtration and Electrophoresis
Objective
The essential goal of the experiment was to separate proteins in a solution based on size in different fractions. The relative protein content for each one fraction was found through the utilization of an amido black-based protein assay. Later in the trial polyacrylamide gel electrophoresis was utilized to separate BSA from hemoglobin.
Methods
I. Gel Filtration and Protein Assay:
1. A slurry of Bio-Gel P-100 beads in water was added to the column (diameter 1.0 cm, length 20 cm) until a height of 10-12 cm was reached.
2. The column was then equilibrated by allowing the liquid level to drop to the top of the column.
3. It was not necessary …show more content…
These fractions were heated at 95° C for 2-3 minutes.
4. The samples were then spun in the centrifuge for 5 seconds.
5. The disc system in electrophoresis consisted of a stacking gel and a resolving gel. The purpose of the stacking gel was to help concentrate the loaded samples in a tight band. The resolving gel was located below the stacking gel and had a smaller pore size allowing the separation of proteins during electrophoresis.
6. In preparation for electrophoresis, 1L of electrode buffer was created by combining 100 ml of 10X electrode buffer with 900 ml of water.
7. The buffer contained the following components:
B-mercaptoethanol, SDS, glycerol, and tracking dye. The purpose of the B-mercaptoethanol and SDS was to break the disulfide bonds and denature the proteins. Glycerol provided added density so that each sample could be loaded through tank buffer, and the tracking dye was used to infer the general locations of the proteins.
8. After lowering the inner cooling core into the buffer chamber, 115 ml of buffer was added to the upper buffer chamber. The rest of the buffer was poured into the lower chambers until the bottom 1 cm of gel was covered. The samples were then loaded, and electrophoresis was performed for 30-40 minutes at 150 …show more content…
The protein hemoglobin was found in portion 7 in both electrophoresis and gel filtration partitions. BSA was likewise effectively known to be in divisions 6, 7, 8, and 9 in the gel filtration examination and by electrophoresis. However, both proteins were dictated by electrophoresis to be in portions 5 through 8, which couldn't be anticipated through the utilization of gel filtration alone. Undoubtedly, there was a mixture of the proteins in a few of the portions, which indicates that the separation was not entirely successful. After studying the gel, it can be seen that the four subunits of hemoglobin all ended up at the 16,000 Daltons marker. This doesn’t mean that these are the actual four subunits of Hemoglobin, they could all be the same but since Beta-Mercaptoethanol separated the Hemoglobin subunits they ended up at 16,000 Daltons marker and not at 64,000 Daltons which is Hemoglobin’s molecular weight. Another band can be seen at 32,000 Daltons marker, which was a dimer of Hemoglobin that didn’t separate. Another band can be seen around the 64,000 Daltons marker, which is where BSA ended up. BSA has a molecular weight of 66,000 Daltons, which makes sense on where the band was observed. Sources of error may have been due to human error in performing the electrophoresis or during the formation of the various samples during gel
Cell Structure and Function
16 October 2014
Gel Filtration and Electrophoresis
Objective
The essential goal of the experiment was to separate proteins in a solution based on size in different fractions. The relative protein content for each one fraction was found through the utilization of an amido black-based protein assay. Later in the trial polyacrylamide gel electrophoresis was utilized to separate BSA from hemoglobin.
Methods
I. Gel Filtration and Protein Assay:
1. A slurry of Bio-Gel P-100 beads in water was added to the column (diameter 1.0 cm, length 20 cm) until a height of 10-12 cm was reached.
2. The column was then equilibrated by allowing the liquid level to drop to the top of the column.
3. It was not necessary …show more content…
These fractions were heated at 95° C for 2-3 minutes.
4. The samples were then spun in the centrifuge for 5 seconds.
5. The disc system in electrophoresis consisted of a stacking gel and a resolving gel. The purpose of the stacking gel was to help concentrate the loaded samples in a tight band. The resolving gel was located below the stacking gel and had a smaller pore size allowing the separation of proteins during electrophoresis.
6. In preparation for electrophoresis, 1L of electrode buffer was created by combining 100 ml of 10X electrode buffer with 900 ml of water.
7. The buffer contained the following components:
B-mercaptoethanol, SDS, glycerol, and tracking dye. The purpose of the B-mercaptoethanol and SDS was to break the disulfide bonds and denature the proteins. Glycerol provided added density so that each sample could be loaded through tank buffer, and the tracking dye was used to infer the general locations of the proteins.
8. After lowering the inner cooling core into the buffer chamber, 115 ml of buffer was added to the upper buffer chamber. The rest of the buffer was poured into the lower chambers until the bottom 1 cm of gel was covered. The samples were then loaded, and electrophoresis was performed for 30-40 minutes at 150 …show more content…
The protein hemoglobin was found in portion 7 in both electrophoresis and gel filtration partitions. BSA was likewise effectively known to be in divisions 6, 7, 8, and 9 in the gel filtration examination and by electrophoresis. However, both proteins were dictated by electrophoresis to be in portions 5 through 8, which couldn't be anticipated through the utilization of gel filtration alone. Undoubtedly, there was a mixture of the proteins in a few of the portions, which indicates that the separation was not entirely successful. After studying the gel, it can be seen that the four subunits of hemoglobin all ended up at the 16,000 Daltons marker. This doesn’t mean that these are the actual four subunits of Hemoglobin, they could all be the same but since Beta-Mercaptoethanol separated the Hemoglobin subunits they ended up at 16,000 Daltons marker and not at 64,000 Daltons which is Hemoglobin’s molecular weight. Another band can be seen at 32,000 Daltons marker, which was a dimer of Hemoglobin that didn’t separate. Another band can be seen around the 64,000 Daltons marker, which is where BSA ended up. BSA has a molecular weight of 66,000 Daltons, which makes sense on where the band was observed. Sources of error may have been due to human error in performing the electrophoresis or during the formation of the various samples during gel