SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
AIM: It is the separation of proteins according to their molecular mass size by moving proteins from the anode to the cathode in the presence of a certain electric field using SDS-PAGE.
INTRODUCTION: SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) is a variant of polyacrylamide gel electrophoresis and is a method in biochemistry that separates charged molecules in mixtures according to their molecular masses in the presence of an electric field[1]. SDS is an important detergent especially used in SDS-PAGE application and investigating the folding/unfolding mechanisms of the protein[1][2]. SDS is an amphipathic detergent and has a lipophilic tail. The hydrophobic tail of the SDS dissolves the hydrophobic regions of the protein, while the sulfate end breaks the ionic bonds after the SDS is attached to the positively charged hydrophobic protein side chains[2]. Thus, the protein loses its secondary and tertiary structure and acquires a linear structure[2]. In the SDS-PAGE procedure, there are 2 types of the gel used[3]. The main component of PAGE is separation gel and consists of SDS, buffer, ammonium persulfate, and TEMED[3]. In the presence of a certain electric field, proteins move from the anode to the cathode, and the speed of this progress varies with the size of the molecular mass. This procedure allows precise separation of proteins on the basis of molecular mass[1][4]. Some chemicals used for SDS; Acrylamide: Hundreds of them come together to form the gel polymer[5]. N, N'-methylene-bis (Acrylamide): Provides gelling and helps to cross-link[5]. TEMED: It is known as tetramethylene diamine and stabilizes free radicals[5]. APS: Known as Ammonium Persulphate and initiates gel formation[5]. Glycerol: It is used to gain the density of the samples[5]. ß-Mercaptoethanol: Causes denaturation[5].
MATERIALS: micropipette and tips, isopropanol, microtube, beaker, microwave, comb, clips , %70 alcohol, diH2O, 10% SDS (100 mL), 10% Ammonium Persulphate (APS) (1 mL), Running Buffer (1 L), Sample Buffer , fixative solution , Coomassie's solution , Destain Solution , Gel Dryer, Whatman filter paper, Seran wrap. - For Separation Gel(7.5%)(20mL): 30% acrylamide/bis, 1.5M Tris-HCl (pH 8.8), 10% SDS, 10% APS, TEMED. - For Stacking Gel(4%)(10mL): 30% acrylamide/bis, 0.5M Tris-HCl (pH 6.8), 10% SDS, 10% APS, TEMED.
METHOD: First, put some water in a 600 mL beaker and microwave to boil. Calculate the desired amount of protein samples for 4 wells (120 µg) and distribute it into a 600 µL microtube. Then, add 2X sample buffer to samples at a 1:1 ratio and place the mixture in the beaker for 5 min. To prepare the separation gel; mix 5,000 µL of 30% acrylamide/bis, 5000 µL of 1.5M Tris-HCl, 200 µL of 10% SDS, 9700 µL of diH2O, and 100 µL of 10% APS in an Erlenmeyer. To prepare the stacking gel; mix 1320 µL of 30% acrylamide/bis, 2520 µL of 0.5M TrisHCl, 100 µL of 10% SDS, 6000 µL of diH2O, and 50 µL of APS in an Erlenmeyer. Then take the separation gel we prepared, add 20 µL of TEMED to it and pour it onto the plate up to the stacking gel limit. Apply a thin layer of isopropanol over the separation gel and wait for the polymerization of the gel. After polymerization, remove the isopropanol gently. Then take the stacking gel, add 20 µL of TEMED to it and apply it onto the separation gel. Place the running buffer in the space between the plates (~ 30 mL). Apply the samples to their wells and add sample buffer to empty wells. When the loading is finished, the lid of the gel tank is closed and the power supply connections are made. Samples are run at 80V through the stacking gel, and at 120V when the run is switched to the separation gel. Follow the run of bromophenol blue, run is finished when blue reaches the lower limit of the gel. Stop the run, open the lid, remove the gel carefully and mark it to remember which side is right and which side is left. Place the gel in fixative solution and incubate for a certain time. Discard the fixative solution, prepare Coomassie's solution, and then apply it to the gel with gentle shaking. Add ammonium sulfate and Coomassie G250 to 400 mL of water until the sulfate dissolves, then add phosphoric acid, and after that methanol slowly. Finally, add the other 400 mL of water. Remove it, prepare the destain solution like that; Mix ethanol, Glacial Acetic Acid, and diH2O in an Erlenmeyer and mix gently. 20. Add 5 mL destain solution and destain for ~1 min with gentle shaking. Discard the destain solution and add 30 mL of destaining solution again. Destain with gentle shaking until the gel is visibly destained (> 2 hr). Discard the destain solution and add 30 mL diH2O and rinse for 5 min with gentle shaking. Dry the gel on the gel dryer at 60°C for 1 hr with a sheet of Whatman filter paper below the gel and a piece of Seran wrap over the gel. Finally, bands are observed under white light in the imaging device.
RESULT: We have successfully concluded our experiment.
figure 1 sample preparation a loading buffer containing SDS beta mercaptoethenol bromophenol blue and glycerol is added to protein samples. When the sample buffer has been added the samples are then 95 degrees celsius for five minutes.
figure 2 gel based on their molecular weight vergil preparation a separating gel solution with pH 8.8 and a stacking gel solutions with pH 6.8 are used and both consist of acrylamide. The gel is produced by polymerization between two glass plates anchored vertically in cassette. the separating gel is poured first.
figure 3 The gel is inserted into a chamber and at risk lysine chloride buffer system with pH 8.3 is poured to allow the conduction of current through the gel SDS is also present in the gel and in running buffer to make sure that once proteins are denatured they stay. Each sample is privoted into its own well in the gel. After sample aplication procedure, an electric fieldis applied across the gell.
figure 4 causing the negatively charged proteins migrate across the gell away from the negative electrode and toward the positive charged. the proteins are detected as blue bands on the clear background. The molecular weight size markrs in a seperate lane in the gel. Can be used the approximate molecular mass of unknown biomolecules by comparing the distance traveled to the marker.
DISCUSSION: SDS-PAGE cannot be used repeatedly as in agarose gel. Because there is physical bonding in agarose gel, but we cannot use it over and over again since there is a chemical bond in SDS PAGE. nucleic acids can be separated by SDS-PAGE. In SDS-PAGE there is only a separation by molecular weight. There is a small amount of 'stacking gel' in a 'running gel' down part. stacking gel allows samples to enter the running gel at the same time. running gel is a little more concentrated and has a higher pH. We can change their concentration by changing the concentration ratio of acrylamide and bisacrylamide. There can be hydrogen bonds and disulfide bonds without applying SDS to proteins. After using SDS, the conformation of the protein becomes primary, since every teraf has a negative charge. hydrophobic regions will not remain in the environment. temperature (60°C) is applied to bring the proteins to the primary structure. This application can be done 1-3-5 minutes. A high concentration gel is used to separate proteins with a down molecular weight and should be low at the voltage used.
REFERENCES:
1. https://tr.wikipedia.org/wiki/SDS-PAGE
2. https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html
3. https://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html
4. https://www.sigmaaldrich.com/technical-documents/articles/biology/sds-page.html
5. https://labakademi.com/sds-page-poliakrilamid-jel-elektroforezi/
