Procedure: Day 1: Buffer preparation First, the buffer was prepared by using the formula as follows: Figure 1: Calculation for prepare 0. 5 M Tris buffer at pH 6. 8 3. 033 g of Tris was weighed and placed in 400 ml beaker. Then, 25 mL of distilled water was added into the beaker that contained Tris. The mixture was dissolved using the stirring rod, and then the magnetic stirring bar was placed in the beaker for further dissolve when measuring the pH. The pH meter was used to measure the solution, and the data was documented at pH 11. 40. This was the starting point. Next, 6M HCl was slowly added into the buffer to make it to pH 6.
Then, the buffer was transferred to a 100ml graduated cylinder, and 25mL of distill water was added to the buffer. Next, the buffer was transferred to 125 mL glass jar and was labeled 0. 5M Tris buffer, pH 6. 8. The top of the flask was sealed with Parafirm and was placed in the refrigerator for next day lab. Other group was prepared other buffers that needed for this experiment. They are 0. 5M Tris pH 6. 8, 1x-running buffer, and transfer buffer. Day 2: Buffer preparation and run the gel and the samples First, the buffer was prepared by using the formula as follows:
Figure 2: Calculation for prepare TBS+NP 40 1x 10mL of TBS +NP 40 30x was measured by 10ml graduated cylinder and placed in a 1000mL Erlenmeyer flask that labeled TBS+ NP 40 1x. Then, 290mL of distilled water was measured by 100 ml graduated cylinder and transferred into the same Erlenmeyer flask that contained the solution. The top of the flask was sealed with Parafirm. Other groups were prepared for other buffers that needed for this experiment. They were TBS 1x, 1x poncheau, 2% gelatin in 1x TBS. Next, the gel-casting unit was assembled by the TA.
The clean and dry electrophoresis equipment was obtained. The short plate was on top of the back plate. The two plates were slide into the casting chamber. The short plate was kept facing front. The thickness of the gap is about 1mm. Then, the TA was put water in each chamber to check for leaking. If it leaks, we have to do it again, if not the water will pour out and dry between plates using Kimwipe or paper towels. Next, The TA was pouring the resolving order according to the order listed in table 1. The Acylamide is hazardous chemical, so she was obtained under the fume hood.
Do not fill all the way. There should be about 1cm of space between the top of the short plate and the resolving gel level (right in the middle of the green plastic bar behind the glass plates). The water was added to the top to prevent from drying. It was allowed to sit for 45 minutes. CUIT Table 1: 12% Resolving gel 1 Water 4. 345mL 21. 5 M Tris Ph 8. 8 2. 5mL 310 % SDS100uL 4 Acylamide 3mL 510% APS (Ammonium per Sufate) 50ul 6 TEMED 5ML After 45 minutes, the TA was poured the stacking gel in order according to table 2 below: Table 2: 4% Stacking Gel 1 Water 3. mL 20. 5 M Tris pH 6. 8 1. 25mL 310 % SDS50UL 4 Acylamide 0. 5mL 510% APS (Ammonium per Sufate) 25uL 6 TEMED 5uL The stacking gel solution should almost fill the remaining space (leave about 2-3 mm between the top of the stacking gel and the top of the short plate).
Then, the TA was inserted the comb, and it was allowed to sit for 45 minutes. After polymerization, the TA was gently removing the glasses with the gel in it from the gel casting apparatus. Then, TA was transferred them both into the electrophoresis unit, the short plate must face inward.
The electrophoresis unit was placed into the electrophoresis bath. The inner chamber was filled with the gel running buffer, and was checked for leaks. Then the TA was added appropriate amount of gel running buffer to the outer chamber. The comb was carefully removed and each loading section of the gel was gently rinsed with the gel running buffer using a 1000 ul pipette. The same gel was run in duplicate. Next, before loading the sample, the TA was centrifuged and then heated the sample at 90°C for 10 minutes to denature the proteins.
The sample was then loaded. There were 10 lanes. The 2nd lane was loaded with the ladder or marker. 5uL of the ladder was measured using micropipette P-10 and added to lane 2. Then, 10 uL of each protein sample was measured using a micropipette P-10 and was added to lane 3,5,6,7,8,9 Lane 3 was cow sample, lane 5 was horse, lane 6 was goat, lane 7 was sheep, lane 8 was donkey, and lane 9 was chicken sample. Nothing was in lane 1,4, and 10. Then the electrophoresis chamber was closed and the gels were run at constant 150V using the power supply for 60 minutes.
Before running the gels, the power supply was set to 150V using the up and down arrow button, then pressed the run button that symbolized with running person on it. If you see the bubble from the bottom it is indicating the gel is running. When finished, disassemble the electrophoresis unit. The “pusher” was gently placed between 2 pieces of glasses to get the gel out in the plastic dish. There were two gels. One gel was stained with the Coomassie blue and the second gel was used to do the transfer for Western Blot.
The first gel was placed in the plastic dish and it was rinsed with distilled water for 3 times, each time it was allowed to soak in distilled water for 3 minutes. This step it helped to remove the SDS in the gel because the SDS will interfere with Coomassie blue and we will not see the band correctly. After the gel was rinsed, in order to visualize the protein, the TA was added the Coomassie blue. Let it stain for 1 hour, then discard the Coomassie blue dye and then add water to remove excess stain and we will see all the bands and the lanes.
The TA was put water to prevent it from drying and let it sit until we come back to take picture. With the second gel, after taking off from the glasses, it was place in the plastic dish and was equilibrated with transfer buffer Tris/ glycine and ready for the “sandwich”. A piece of nitrocellulose membrane and pieces of blot paper and sponges was prepared. All the “sandwich” material was soaked in the transfer buffer for 10 minutes. The plastic cassette was obtained and the black of the cassette was facing down.
Make the sandwich as follow: 2 sponges were placed in the bottom of the cassette, next was the blot paper (thick paper for support). Then, the SDS-PAGE gel was placed on top of the blot paper with the flip the orientation left to right, the nitrocellulose membrane was placed next, then the blot paper, and last was 2 sponges. The cassette was closed. Before close the cassette, a test tube was rolled over the top of the sponges’ layer of the sandwich to ensure that the membrane and the gel are pressed against each other without any air bubbles that will interfere with the transfer.
The sandwich was then inserted into the electrophoresis chamber (the black part of cassette goes with the black of the electrophoresis chamber). Then, the electrophoresis chamber was filled up with the transfer buffer in one side and one side was contained ice. The other side was the cassette. It was run at constant 100V usi the power supply for 60 minutes. When finished, the cassette was taken out and the nitrocellulose was taken out also. Then, the poncheau was added and let it was allowed to sit for a couple minutes. The poncheau stain is not specific so it will stain every protein on the nitrocellulose.
The purpose of this is to verify if the transfer work. Then, rinse with water because the poncheau soluble in water. The next step is the blocking, 2% gelatin was added to the nitrocellulose and it was allowed to sit overnight, then the gelatin was removed to perform next step. Day 3: Add antibody and develop blot 1:100 primary antibody was prepared as follows: Figure 3: The calculation of diluting antibody to cow albumin 1:100 in 7mL 70ul of the primary antibody and 6. 97mL of 2% gelatin was added into the nitrocellulose. The primary antibody recognizes a specific amino-acid sequence of a particular protein.
In this case, the antibody was anti-cow albumin. Then, it was incubated at 37°C for 30 minutes. After incubated, it was rinsed with TBS +NP 40 1x for 4 times. Each time was 5 minutes. This step was to wash the unbound antibodies. Then, it was rinsed two times with TBS 1x for 5 minutes to remove the soap. The blot was then developed using the color development. 7mL of chlornaptol and 1 l H202 was measured and added into the nitrocellulose and it was allowed to sit for 10-15 minutes for the color to develop. Then, the light purple color was presented.
The picture was taken and recoded in result. Result: Figure 4: The picture of SDS-PAGE gel showing the presence of serum albumen in animals such as cow, horse, goat, sheep and donkey and chicken. Lane 4 supposes to have nothing but it was showed blue. It blue maybe because the sample of cow or horse was somehow get in lane 4. Figure 5: The picture shows the transferred proteins from the gel to the nitrocellulose and was stained by Pancheau to test if the transfer works. Figure 6: The picture of the western blot result after performed the color development.