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…
to equilibrate the column since the beads were already in the buffer.
4. The sample (a mixture of 2 mg/ml Blue Dextran, 2 µl yellow food coloring, 2 mg/ml hemoglobin, and 2 mg/ml BSA) was then added to the column.
5. The fraction samples were collected as the sample flowed through the column in volumes of 0.5 ml (4 drops). When the liquid level fell to the top of the beads, 1 ml of buffer was added to the column, and it would be allowed to flow into the column.
6. The column would be stopped, and buffer would be added until the column was again full.
7. The column would again be allowed to resume collecting the next fraction.
8. To assay the proteins, 1 µl of each fraction was spotted on a nitrocellulose filter.
9. The filter was allowed to dry and was then incubated in 50 % methanol-10% acetic acid for 5 minute.
10. The filter was then incubated in amido black for 5 minutes. The purpose of amido black was to bind to the free amino groups of protein and make them visible.
11. The filter was then rinsed several times with 50% methanol-10% acetic acid. The intensity of each spot represented the amount of protein in the sample.
12. We kept tubes 5-12 (8 Eppendorf tubes) to be used for SDS-PAGE for next week.
II. SDS-PAGE
1. Fractions that ranged from the starting elution of Blue Dextran to the starting elution of potassium chromate were assayed.
2. 25 µl of each fraction was mixed with 25 microliters of 2x sample buffer.
3.
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…
V.
9. The gel was then placed in a 1/4 to 3/8 inch stain box.
10. After 15 minutes, pour staining solution into used stain bottle.
11. Replace the stain with 1/4 to 3/8 of destaining solution until next week.
Results
I. Gel Filtration and Protein Assay
Table 1. Color, Volume, and Protein Content for each Fraction
Fraction Color Volume (ml) Relative Protein Content Compound Determined
1 Clear 0.5 None None
2 Clear 0.5 None None
3 Clear 0.5 None None
4 Clear 0.5 None None
5 Clear 0.5 Medium dot, moderate protein content BSA
6 Blue 0.5 Slight dot, low protein content Blue Dextran and protein
7 Slight Blue 0.5 Slight dot, low protein content Blue Dextran and protein
8 Slight brown 0.5 Heavy dot, high protein content Hemoglobin
9 Clear 0.5 Medium dot, moderate protein content BSA
10 Clear 0.5 Slight dot, low protein content BSA
11 Clear 0.5 None None
12 Clear 0.5 None None
13 Clear 0.5 None None
14 Clear 0.5 None None
15 Clear 0.5 None None
16 Slight Yellow 0.5 None Sodium chromate
17 Slight Yellow 0.5 None Sodium chromate
18 Slight Yellow 0.5 None Sodium chromate
19 Yellow 0.5 None Sodium chromate
20 Yellow 0.5 None Sodium chromate
21 Slight Yellow 0.5 None Sodium chromate
22 Clear 0.5 None None Height of column = 6.1 cm
Diameter = 1 cm
Radius = 0.5 cm
Calculations
Accessible volume (Vo +Vi) = volume of sodium chromate = 19 x 0.5 ml = 9.5 ml
Void volume (Vo) = volume of Blue Dextran = 6 x 0.5 ml =3.0 ml
Included volume (Vi) = accessible volume – void volume = 9.5 -3.0 =6.5 ml
Inert Volume (Vg) = height- accessible volume = πr2h – (Vo + Vi) = (π)(0.5 cm)2(6.1) – (19)(0.5 ml) = 4.71 ml
Volume diluted of sodium chromate = Vf-Vi = (21 x 0.5 ml) – (16 x 0.5) = 2.5 ml
Volume diluted of Blue Dextran = Vf-Vi = (7 x 0.5 ml) – (6 x 0.5) = 0.5 ml
Volume diluted of hemoglobin = Vf-Vi = (8 x 0.5 ml) – (7 x 0.5) = 0.5 ml
Volume diluted of BSA = Vf-Vi = (10 x 0.5 ml) – (5 x 0.5) = 2.5 ml
II. SDS-PAGE
Table 2. Fraction Volumes used for Electrophoresis
Well # Sample Sample Volume (μl)
1 Standard Marker Mix 10
2 Fraction 6 30
3 Fraction 7 30
4 Fraction 8 30
5 Fraction 9 30
6 Fraction 10 30
7 Fraction 11 30
8
Fraction 12 30
9 Fraction 13 30
10 Standard Marker Mix 10
Discussion In the introductory piece of the trial, gel filtration was utilized to partition compounds in a sample. By watching the distinctive colors of the parts divided, the accessible volume, inert volume, void volume, and included volume could be calculated for the column. The accessible volume was discovered to be 9.5 ml. This was resolved through the volume eluted by the compound with the smallest size, which is sodium chromate. Sodium chromate was known to have a yellow shade, and fraction 19 was found to have the strongest yellow color and with no doubt, fraction 19 would have the highest concentration of sodium chromate. At this fraction, the eluted volume was 9.5 ml, which makes the accessible volume 9.5 ml. To calculate the void volume we had to look for a compound with the largest size, which is Blue Dextran. Fraction 6 was found to have the strongest blue shade, which implies that Blue Dextran was most packed in that fraction. The void volume was discovered to be 3 ml. The included volume (volume of all the pores) was found through subtracting the void volume from the accessible volume. This was discovered to be 6.5 ml. The inert volume, which was found by subtracting the accessible volume from the height of the column, was discovered to be 4.71 ml. The volume diluted for every substance was dictated by subtracting the final volume from the initial volume for every substance. The values obtained for sodium chromate, Blue Dextran, hemoglobin, and BSA were 2.5 ml, 0.5 ml, 0.5ml, and 2.5 ml correspondingly. There was no discernible patterns or criticalness found in these numbers. Hemoglobin, perceived as being the brown in color, was found in the seventh eluted portion. The molecular weight of hemoglobin, 64 kDa1, which demonstrates that it ought to be for first to elute since it is large in size, which implies it can go in the between all of the pores without having to go through them, hence elutes quicker. BSA is believed to have a molecular weight of 66kDa2. Because of its clear shade, BSA could have eluted in any of the portions that were resolved to have protein (portions 5 through 10). Be that as it may because of its molecular weight, it is theorized to have eluted around the time hemoglobin eluted, which is around the seventh portion. The gel filtration column was seen to have divided with precision the Blue Dextran (portions 6 and 7) and the sodium chromate (portions 16 through 21). The protein Hemoglobin, which is known to have a brown shade, was found in portion 7. Through the utilization of the protein assay, we discovered that the protein BSA could be found in any of the portions from 5 through 10. However, the accuracy of the protein separation can only be determined after performing SDS-PAGE. The second piece of the lab included utilizing polyacrylamide gel electrophoresis; P.A.G.E. was performed to differentiate proteins on the premises of molecular size. Samples that incorporated an arrangement of protein markers and our portions were loaded into wells. After the allotted time had been given for the gel to run its cycle, the gel was then stained then later on de-stained for further examination. The viability in differentiating the BSA and hemoglobin utilizing gel filtration was not very conclusive.
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
filtration.
References
1 National Center for Biotechnology Information. (n.d.) Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23685348
2 Sigma-Aldrich. (n.d.). Retrieved from http://www.sigmaaldrich.com/catalog/product/sigma/a8531?lang=en®ion=US
Sheffield, J. 2011. Biology 3096 Lab Manual. Temple University.
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…
to equilibrate the column since the beads were already in the buffer.
4. The sample (a mixture of 2 mg/ml Blue Dextran, 2 µl yellow food coloring, 2 mg/ml hemoglobin, and 2 mg/ml BSA) was then added to the column.
5. The fraction samples were collected as the sample flowed through the column in volumes of 0.5 ml (4 drops). When the liquid level fell to the top of the beads, 1 ml of buffer was added to the column, and it would be allowed to flow into the column.
6. The column would be stopped, and buffer would be added until the column was again full.
7. The column would again be allowed to resume collecting the next fraction.
8. To assay the proteins, 1 µl of each fraction was spotted on a nitrocellulose filter.
9. The filter was allowed to dry and was then incubated in 50 % methanol-10% acetic acid for 5 minute.
10. The filter was then incubated in amido black for 5 minutes. The purpose of amido black was to bind to the free amino groups of protein and make them visible.
11. The filter was then rinsed several times with 50% methanol-10% acetic acid. The intensity of each spot represented the amount of protein in the sample.
12. We kept tubes 5-12 (8 Eppendorf tubes) to be used for SDS-PAGE for next week.
II. SDS-PAGE
1. Fractions that ranged from the starting elution of Blue Dextran to the starting elution of potassium chromate were assayed.
2. 25 µl of each fraction was mixed with 25 microliters of 2x sample buffer.
3.
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…
V.
9. The gel was then placed in a 1/4 to 3/8 inch stain box.
10. After 15 minutes, pour staining solution into used stain bottle.
11. Replace the stain with 1/4 to 3/8 of destaining solution until next week.
Results
I. Gel Filtration and Protein Assay
Table 1. Color, Volume, and Protein Content for each Fraction
Fraction Color Volume (ml) Relative Protein Content Compound Determined
1 Clear 0.5 None None
2 Clear 0.5 None None
3 Clear 0.5 None None
4 Clear 0.5 None None
5 Clear 0.5 Medium dot, moderate protein content BSA
6 Blue 0.5 Slight dot, low protein content Blue Dextran and protein
7 Slight Blue 0.5 Slight dot, low protein content Blue Dextran and protein
8 Slight brown 0.5 Heavy dot, high protein content Hemoglobin
9 Clear 0.5 Medium dot, moderate protein content BSA
10 Clear 0.5 Slight dot, low protein content BSA
11 Clear 0.5 None None
12 Clear 0.5 None None
13 Clear 0.5 None None
14 Clear 0.5 None None
15 Clear 0.5 None None
16 Slight Yellow 0.5 None Sodium chromate
17 Slight Yellow 0.5 None Sodium chromate
18 Slight Yellow 0.5 None Sodium chromate
19 Yellow 0.5 None Sodium chromate
20 Yellow 0.5 None Sodium chromate
21 Slight Yellow 0.5 None Sodium chromate
22 Clear 0.5 None None Height of column = 6.1 cm
Diameter = 1 cm
Radius = 0.5 cm
Calculations
Accessible volume (Vo +Vi) = volume of sodium chromate = 19 x 0.5 ml = 9.5 ml
Void volume (Vo) = volume of Blue Dextran = 6 x 0.5 ml =3.0 ml
Included volume (Vi) = accessible volume – void volume = 9.5 -3.0 =6.5 ml
Inert Volume (Vg) = height- accessible volume = πr2h – (Vo + Vi) = (π)(0.5 cm)2(6.1) – (19)(0.5 ml) = 4.71 ml
Volume diluted of sodium chromate = Vf-Vi = (21 x 0.5 ml) – (16 x 0.5) = 2.5 ml
Volume diluted of Blue Dextran = Vf-Vi = (7 x 0.5 ml) – (6 x 0.5) = 0.5 ml
Volume diluted of hemoglobin = Vf-Vi = (8 x 0.5 ml) – (7 x 0.5) = 0.5 ml
Volume diluted of BSA = Vf-Vi = (10 x 0.5 ml) – (5 x 0.5) = 2.5 ml
II. SDS-PAGE
Table 2. Fraction Volumes used for Electrophoresis
Well # Sample Sample Volume (μl)
1 Standard Marker Mix 10
2 Fraction 6 30
3 Fraction 7 30
4 Fraction 8 30
5 Fraction 9 30
6 Fraction 10 30
7 Fraction 11 30
8
Fraction 12 30
9 Fraction 13 30
10 Standard Marker Mix 10
Discussion In the introductory piece of the trial, gel filtration was utilized to partition compounds in a sample. By watching the distinctive colors of the parts divided, the accessible volume, inert volume, void volume, and included volume could be calculated for the column. The accessible volume was discovered to be 9.5 ml. This was resolved through the volume eluted by the compound with the smallest size, which is sodium chromate. Sodium chromate was known to have a yellow shade, and fraction 19 was found to have the strongest yellow color and with no doubt, fraction 19 would have the highest concentration of sodium chromate. At this fraction, the eluted volume was 9.5 ml, which makes the accessible volume 9.5 ml. To calculate the void volume we had to look for a compound with the largest size, which is Blue Dextran. Fraction 6 was found to have the strongest blue shade, which implies that Blue Dextran was most packed in that fraction. The void volume was discovered to be 3 ml. The included volume (volume of all the pores) was found through subtracting the void volume from the accessible volume. This was discovered to be 6.5 ml. The inert volume, which was found by subtracting the accessible volume from the height of the column, was discovered to be 4.71 ml. The volume diluted for every substance was dictated by subtracting the final volume from the initial volume for every substance. The values obtained for sodium chromate, Blue Dextran, hemoglobin, and BSA were 2.5 ml, 0.5 ml, 0.5ml, and 2.5 ml correspondingly. There was no discernible patterns or criticalness found in these numbers. Hemoglobin, perceived as being the brown in color, was found in the seventh eluted portion. The molecular weight of hemoglobin, 64 kDa1, which demonstrates that it ought to be for first to elute since it is large in size, which implies it can go in the between all of the pores without having to go through them, hence elutes quicker. BSA is believed to have a molecular weight of 66kDa2. Because of its clear shade, BSA could have eluted in any of the portions that were resolved to have protein (portions 5 through 10). Be that as it may because of its molecular weight, it is theorized to have eluted around the time hemoglobin eluted, which is around the seventh portion. The gel filtration column was seen to have divided with precision the Blue Dextran (portions 6 and 7) and the sodium chromate (portions 16 through 21). The protein Hemoglobin, which is known to have a brown shade, was found in portion 7. Through the utilization of the protein assay, we discovered that the protein BSA could be found in any of the portions from 5 through 10. However, the accuracy of the protein separation can only be determined after performing SDS-PAGE. The second piece of the lab included utilizing polyacrylamide gel electrophoresis; P.A.G.E. was performed to differentiate proteins on the premises of molecular size. Samples that incorporated an arrangement of protein markers and our portions were loaded into wells. After the allotted time had been given for the gel to run its cycle, the gel was then stained then later on de-stained for further examination. The viability in differentiating the BSA and hemoglobin utilizing gel filtration was not very conclusive.
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
filtration.
References
1 National Center for Biotechnology Information. (n.d.) Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23685348
2 Sigma-Aldrich. (n.d.). Retrieved from http://www.sigmaaldrich.com/catalog/product/sigma/a8531?lang=en®ion=US
Sheffield, J. 2011. Biology 3096 Lab Manual. Temple University.