Samples<\/h2>\n<\/span>\n\n\nThe sample preparation is crucial in the case of the SDS-PAGE process. But to make it more simple, let’s go through an example by which it will be easier. <\/p>\n\n\n\n
“From the clinics, we got 4 samples from four different patients. We know that the disease for what we are looking is characterized by a high level of p53 for example. About the p53 we know that the molecular weight of the protein is exactly 53 kDa. We need to run the samples and detect it.<\/em>“<\/p>\n\n\n\nThe crude sample from the patients contain hundreds of different sized and shaped proteins. In order to find our protein of interest, we need to cast a gel, which is capable to separate the proteins based on the size. <\/p>\n\n\n\n
<\/p>\n\n\n<\/span>\nGEL CASTING<\/h2>\n<\/span>\n\n\nThe basic component of the gel is acrylamide. This molecule is highly toxic, it is accumulating in the brain, thus the tissue damages slowly. The same molecule will appear in the oil that we are using for the fried dishes. Even more, we use the same oil even more acrylamide is generating. (Please do not use more than 2 times the same oil)<\/p>\n\n\n\n
The gel has two separate parts the “bottom” and the “top” part. <\/p>\n\n\n\n
The bottom part of the gel is the so-called separating gel. This is the bigger part of the whole structure, while the top is the sample collection gel. Every time we need to prepare two mixtures, one for the bottom and one for the top gels. In this mixture not only the acrylamide is there but also a buffer, a catalyzation and a crosslinker. <\/p>\n\n\n\n
To have a stable structure of the gel, we need to generate or create a “net” for the proteins. This net is made by acrylamide polymers. To crosslink and activate the acrylamide, we are using the so-called Ammonium persulphate (APS) and the TEMED (tetramethyl-diethyl-amine) (Figure 1).<\/p>\n\n\n\n![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/07\/sds-1024x739.jpg\")
<\/a>Figure 1. Structure of the SDS gel<\/strong><\/figcaption><\/figure>\n\n\n\n
By so far if we put together all these components, we got a “native” gel. But as you read before SDS we need to use. <\/p>\n\n\n\n
SDS gives a total net negative charge for all of the proteins. And we need to put it into the mix as well (Table 1).<\/p>\n\n\n\n<\/td> WATER<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> TRIS BUFFER<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> SDS<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> APS (Ammonium persulphate)<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> TEMED (Tetramethyl-diethyl-amine)<\/td> <\/td> <\/td> <\/td> <\/td><\/tr><\/tbody><\/table>Table 1. <\/figcaption><\/figure>\n\n\n\nAfter we put it together and mix well we got a totally transparent solution, which is highly liquid. By the time because the APS catalyses the reaction of the crosslink formation, the gel structure slowly developed. The separating gel (bottom) takes 10-25 minutes. It depends on how much acrylamide is in it. The content of the acrylamide is given by percentage: 6%, 8%, 10%, 12%, 15%. Even higher the percentage even solid the gel would be.<\/p>\n\n\n\n
The upper gel needs to be formed by generating wells in it, For that we use usually a plastic “comb”. After the upper the so-called stacking gel is polymerized we need to prepare the samples for running.<\/p>\n\n\n<\/span>\nSample preparation<\/h2>\n<\/span>\n\n\nSamples have a lot of different proteins, in their native structure. Which could be a problem in the case of the running. the reason we are using the SDS in the gel, and also in the sample buffer, is to give a total net charge to the proteins. This buffer is called a denaturation buffer with a blue colour. The colour comes from the Bromphenol-blue dye. This dye also helps to visualize where the samples are actually running in the gel.<\/p>\n\n\n\n
After we dissolve the samples in the denaturation buffer we need to boil the samples. This process takes 10 minutes at 99 celcius resulted in linearized negatively charged proteins. After that, we need to load the samples into the precast gels (Figure 2). <\/p>\n\n\n\n![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/07\/sds2-1024x399.jpg\")
<\/a>Figure 2. From left to right: sample loading, running starts, running further<\/strong><\/figcaption><\/figure>\n\n\n<\/span>\nRunning<\/h2>\n<\/span>\n\n\nBased on the electrophoresis principle if we give voltage \/electricity to the system, the negatively charged proteins by the SDS are going to migrate into the gel towards the positive side. Meanwhile, the blue colour forms the front line, which represents the current position of the dye and the smallest proteins.<\/p>\n\n\n\n
For the running, we need to use pre-coloured protein ladders to monitor where is the running going. Based on the current protein of interest we can exactly expect for example the p53 is still in the gel or not.<\/p>\n\n\n\n
The running usually goes in two phases :<\/p>\n\n\n\n
- 1-15 minutes 120 V<\/li>
- 15 minutes up to 75-85 minutes 150 V<\/li><\/ol>\n\n\n\n
After the running is done, we need to separate the running cassette to reach the gel.<\/p>\n\n\n\n
The gel can be stained with Coomassie blue for the proteins. which give us a piece of semi information about how many proteins are there in our sample. however, it won’t give and exact information about the protein of interest by the lack of specific signs (Figure 3).<\/p>\n\n\n\n![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/07\/Comassie-830x1024.jpg\")
<\/a>Figure 3. Coomassie-stained gel represents the proteins in the sample after SDS-running<\/figcaption><\/figure>\n<\/span>
The crude sample from the patients contain hundreds of different sized and shaped proteins. In order to find our protein of interest, we need to cast a gel, which is capable to separate the proteins based on the size. <\/p>\n\n\n\n
<\/p>\n\n\n<\/span>\n The basic component of the gel is acrylamide. This molecule is highly toxic, it is accumulating in the brain, thus the tissue damages slowly. The same molecule will appear in the oil that we are using for the fried dishes. Even more, we use the same oil even more acrylamide is generating. (Please do not use more than 2 times the same oil)<\/p>\n\n\n\n The gel has two separate parts the “bottom” and the “top” part. <\/p>\n\n\n\n The bottom part of the gel is the so-called separating gel. This is the bigger part of the whole structure, while the top is the sample collection gel. Every time we need to prepare two mixtures, one for the bottom and one for the top gels. In this mixture not only the acrylamide is there but also a buffer, a catalyzation and a crosslinker. <\/p>\n\n\n\n To have a stable structure of the gel, we need to generate or create a “net” for the proteins. This net is made by acrylamide polymers. To crosslink and activate the acrylamide, we are using the so-called Ammonium persulphate (APS) and the TEMED (tetramethyl-diethyl-amine) (Figure 1).<\/p>\n\n\n\n SDS gives a total net negative charge for all of the proteins. And we need to put it into the mix as well (Table 1).<\/p>\n\n\n\n After we put it together and mix well we got a totally transparent solution, which is highly liquid. By the time because the APS catalyses the reaction of the crosslink formation, the gel structure slowly developed. The separating gel (bottom) takes 10-25 minutes. It depends on how much acrylamide is in it. The content of the acrylamide is given by percentage: 6%, 8%, 10%, 12%, 15%. Even higher the percentage even solid the gel would be.<\/p>\n\n\n\n The upper gel needs to be formed by generating wells in it, For that we use usually a plastic “comb”. After the upper the so-called stacking gel is polymerized we need to prepare the samples for running.<\/p>\n\n\n<\/span>\n Samples have a lot of different proteins, in their native structure. Which could be a problem in the case of the running. the reason we are using the SDS in the gel, and also in the sample buffer, is to give a total net charge to the proteins. This buffer is called a denaturation buffer with a blue colour. The colour comes from the Bromphenol-blue dye. This dye also helps to visualize where the samples are actually running in the gel.<\/p>\n\n\n\n After we dissolve the samples in the denaturation buffer we need to boil the samples. This process takes 10 minutes at 99 celcius resulted in linearized negatively charged proteins. After that, we need to load the samples into the precast gels (Figure 2). <\/p>\n\n\n\n Based on the electrophoresis principle if we give voltage \/electricity to the system, the negatively charged proteins by the SDS are going to migrate into the gel towards the positive side. Meanwhile, the blue colour forms the front line, which represents the current position of the dye and the smallest proteins.<\/p>\n\n\n\n For the running, we need to use pre-coloured protein ladders to monitor where is the running going. Based on the current protein of interest we can exactly expect for example the p53 is still in the gel or not.<\/p>\n\n\n\n The running usually goes in two phases :<\/p>\n\n\n\n After the running is done, we need to separate the running cassette to reach the gel.<\/p>\n\n\n\n The gel can be stained with Coomassie blue for the proteins. which give us a piece of semi information about how many proteins are there in our sample. however, it won’t give and exact information about the protein of interest by the lack of specific signs (Figure 3).<\/p>\n\n\n\nGEL CASTING<\/h2>\n<\/span>\n\n\n
![\"\"](\"https:\/\/meddists.com\/learn\/wp-content\/uploads\/2021\/07\/sds-scaled.jpg\")
<\/a>
By so far if we put together all these components, we got a “native” gel. But as you read before SDS we need to use. <\/p>\n\n\n\n<\/td> WATER<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> TRIS BUFFER<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> SDS<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> APS (Ammonium persulphate)<\/td> <\/td> <\/td> <\/td> <\/td><\/tr> <\/td> TEMED (Tetramethyl-diethyl-amine)<\/td> <\/td> <\/td> <\/td> <\/td><\/tr><\/tbody><\/table> Sample preparation<\/h2>\n<\/span>\n\n\n
<\/a>Running<\/h2>\n<\/span>\n\n\n
<\/a>