SDS-PAGE – Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (PAGE)

Introduction of SDS-PAGE

SDS-PAGE (Sodium dodecyl Sulfate) polyacrylamide gel electrophoresis is one of the methods employed in genetics, biochemistry and molecular biology for the separation of proteins based on the molecular mass of their proteins. Proteins’ electrophoretic mobility is dependent on their size. The goal of SDS-PAGE is the separation of proteins based on their dimensions.

Since proteins are amphoteric substances and have a net charge, their charges is able to be measured according to their pH that the media in which they’re suspended. So, at a certain pH and in non-denaturing conditions the electrophoretic separation between protein is determined by the dimensions and charges of the molecules. Since proteins are heavy molecular weight compounds, it requires porous gels in order to be separated.

Polyacrylamide gels can be used to separate proteins by size since the gels are porous. This kit allows students to master the art of SDS-PAGE. 

What is SDS PAGE?

SDS-PAGE is one of the most widely utilized method of qualitatively analyzing any protein-based mixture, to check the purity of the protein and to determine the molecular mass of proteins. It’s based on the sorting of proteins in accordance to their size and then finding them through binding to dye.

For the separation of different proteins that have different sizes and shapes They must first be denatured, so that the proteins do not contain an additional, secondary, or quternary structure. The sodium dodecylsulphate (SDS) is an anionic detergent that denatures proteins through “wrapping around” the polypeptide backbone. SDS removes all proteins back to their original structure. SDS gives negative charges to the polypeptide, in proportion of its length. SDS treatment is distinguished by two key aspects:

1. All proteins retain only their primary structure.
2. All proteins have a large amount of negative charge.

Polyacrylamide is the ideal gel to create this kind of environment. Polyacrylamide is synthetic gel that is transparent, thermo-stable and thermo-stable. It is sturdy and chemically inert. It can be created with a range of pore sizes. It is able to endure high voltage gradients and can be used for various destaining and staining methods and is digestible to separate fractions, or dried for autoradiography or permanent recording. A polymer gel is composed from acrylamide monomers. The proteins are pushed through the gel via electrophoresis and this whole procedure is known as the Polyacrylamide Gel Electrophoresis (PAGE).

Two layers are made of gel specifically, spacer gel, or stacking gel and Separating or resolving gel.

1. Stacking or spacer gel: The gel that is used for stacking contains huge pores made of polyacrylamide (generally at 5%). The gel is made with Tris buffer with pH 6.8 which is approximately two pH units higher than the pH in the buffer for electrophoresis. The gel is formed on top of the separation gel.
2. Separating or resolving gel: The gel used for separation has small pores made of polyacrylamide (5-30 5-30%). The Tris buffer used is pH 8.8. This gel is where macromolecules are separated according to the size of their molecules.

Objective

• To know how to prepare Sodium Dodecyl Sulphate Polyacrylamide Gel (SDS-PAGE)
• To determine the purity of protein by SDS-PAGE as well as
• To determine the molecular mass of protein purified by SDS-PAGE.

Principle of SDS-PAGE

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is carried out in a discontinuous buffer system wherein the reservoir buffer is of a different pH and ionic strength from the buffer used to cast the gel. The SDS polypeptide complexes in the sample applied to the gel are swept along by a moving boundary created when an electric current is passed between the electrodes. After migration through the stacking gel of high porosity, complexes get deposited in a very thin zone on the surface of the resolving gel. On further electrophoresis, polypeptides get resolved based on their size in the resolving gel.

The materials used in SDS-PAGE and their roles

• Tris: Tris is utilized as a buffer because it is an innocuous chemical to the majority of proteins. Its pKa value is 8.3 in 20oC and is considered to be a suitable buffer within the pH range of 7.0 to 9.0.
• Acrylamide: Acrylamide is a white powder, when it dissolves into water, autopolymerisation happens. It’s a slow and spontaneous process in which the acrylamide molecules bind in a head-on-tail fashion. However, in the absence of free radicals in the systems, acrylamide monomers are transformed into a state of free radicals. Monomers that are activated polymerise quickly and create long chains of polymers. This type of reaction is referred to in the field of Vinyladdition polymerisation.
• Bisacrylamide (N,N’-Methylenebisacrylamide): Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels. Chemically, it is composed of two acrylamide molecules bonded head-to-head at their non-reactive ends.
• Sodium Dodecyl Sulphate (SDS): SDS is the most widely used denaturing agent to reduce native proteins into polypeptides. When a mixture of proteins is heated to temperatures of 100 degrees Celsius in the presence of SDS the detergent is wrapped around the polypeptide’s backbone. This binds polypeptides with an unchanging proportion of 1.4 grams per gram of polypeptide. This process causes the charge of polypeptides are negligible they are compared with the negative charges generated by SDS. Therefore, polypeptides treated transform into a rod-like structure with a uniform charge density with the equal negative charge per.
• Ammonium Persulphate (APS): The APS chemical is a catalyst to gel formation.
• 6. N, N, N’, N’-tetramethylethylenediamine (TEMED): Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium persulfate and the quaternary amine, N,N,N’,N’- tetramethylethylenediamine (TEMED).

Requirement for SDS PAGE

• Acrylamide/Bisacrylamide Solution 30% (29:1)
• 2.5X Tris-SDS Buffer (pH 8.8) 65 ml 125 ml 2-8o
• 5X Tris-SDS Buffer (pH 6.8)
• Prestained Protein Ladder
• 5X Tris-Glycine-SDS Gel Running Buffer 
• 5X Sample Loading Buffer
• Protein Sample 1
• Protein Sample 2
• Staining solution
• Destaining solution
• Ammonium persulphate (APS)
• Tetramethylethylenediamine (TEMED)
• Agarose 

Other Material Requireds;

• Glass wares: Conical flask, Measuring cylinder, Beaker
• Reagents: Distilled water
• Other requirements: Protein Electrophoresis apparatus, Micropipettes, Tips, Microwave/Burner/Hotplate 

SDS or sodium dodecyl sulphate

SDS, or sodium dodecylsulphate is an anionic detergent which is able to bind tightly to proteins, leading to their denature. In the presence of excessive SDS approximately 1.4 grams of the detergent will bind to every one gram of protein, giving the protein the same negative charge per mass. In the end, SDS-protein complexes move toward the anode during electrophoresis , and due to the molecular sieving characteristics in the polyacrylamide gel are separated according to their weights in molecular units. The principle behind this method is to separate proteins on the basis of size differences when applying standard proteins with known molecular weights on the same gel as an unknown proteins, the molecular weight of the protein unknown can be measured. Mobility of proteins in SDS electrophoresis gels is defined in terms of relative mobility (R f) relative to the dye used for tracking bromophenol blue.

Rf values of markers for proteins that are known in size are utilized to produce an atypical curve by placing the molecular weights in relation to each Rf value on an semi-log graph. The molecular mass of the unknown protein is then extrapolated from the Rf value.

How does SDS PAGE work?

When the voltage is applied, the chloride ions within the sample buffer and in the stacking gel rapidly move towards that pole of positive charge, and form an edge leading to the moving Ion Front. Glycine molecules possess very little charge in the gel stacking and therefore, they move to the back of the front of ions. The difference in glycine and chloride mobility results in a significant voltage gradient within the gel, which sweeps across negative charged complexes of protein and SDS. The huge pores of the stacking gel offer minimal resistance to the movement of protein-SDS complexes. They are then able to “stack up” into a extremely concentrated area at the intersection of the stacking and running gels (right). ProteinSDS complexes are concentrated near the interface until gradually moving glycine molecules reach the boundaries of the gels.

Significant changes take place as glycine ions move into the gel. They change the pH of the running gel is close to the pKa of amino groups of glycine, which means that an important portion of Glycine molecules acquire negative charges. Glycine molecules with negative charges start to travel at the same speed as the chloride ions thus removing the voltage gap that controlled the movement of proteins through the bed of the. The pores of the run gels are smaller than the gel used for stacking, therefore the pores provide resistance to the movement of proteins. Proteins start to migrate at various rate due to the sieving properties of gel. Smaller protein-SDS complexes move faster than complexes with larger amounts of proteinSDS (right). Within a specific range defined from the degree of porosity the rate of migration of proteins in the running gel is proportional to the logarithms of the MW.

SDS PAGE Protocol

1. Make sure that glass panels are secured to the unit, along with the spacers positioned on two edges.
2. Make 1 % agarose (0.05g within 5ml distillated water). Boil until the agarose dissolves and then pour an even layer of horizontal agarose at the bottom of the plates to secure the plate. Allow it to solidify it to cool for 5-10 mins.

Preparation of 12% Separating Gel

For the preparation of separating gel include the ingredients as follows:

Ingredients	Amount
30% Acrylamide-bisacrylamide Solution	6 ml
Distilled water	3 ml
2.5X Tris-SDS Buffer (pH 6.8)	6 ml
10% APS Solution	125 ul
TEMED	18 ul

Place the gel between the plates and let it set for about one hour. After the gel has cooled, immediately pour it onto the plates.

Poured, add distilled water until it is level the gel. After about an hour, remove all the liquid by flipping the casting.

Preparation of 5% Stacking Gel

To prepare the stacking gel mix the ingredients in the following order:

Ingredients	Amount
30% Acrylamide-bisacrylamide Solution	1.3 ml
Distilled water	5.1 ml
5X Tris-SDS Buffer (pH 6.8)	1.6 ml
10% APS Solution	75 ul
TEMED	10 ul

After the addition of TEMED thoroughly mix all ingredients by swirling the beaker. Place the stacking gel on the top of the separating gel. Then, immediately, place the comb, making sure to avoid air bubbles. It should be allowed to set for about 30 minutes.

Important: Acrylamide is a potential neurotoxin, and must be handled with extreme attention. Always wear a face-mask and wear gloves.

3. Place 1X Tris-Glycine-SDS Gel Running Buffer into the unit in a way that it connects the two electrodes and consequently completes that flow. Take the comb out of the stacking Gel cautiously.
4. Sample Preparation: Take 2 tubes for protein samples. Label them accordingly. You should take 20 ml of each sample from the tube and add 5ml of the 5X sample loading buffer it. The tubes that contain Protein Samples at 100oC in the bath of boiling water. Don’t boil the tube that has PrestainedProtein Ladder.
5. Take 5ml of the Prestained Protein Ladder and 20ml of the samples right after the heat treatment of the wells made by the comb within the Stacking Gel.
6. The power cable should be connected to an electrophoretic power source according to the standard protocols: Red-Anode as well as Black-Cathode. The electrophoresis process is 100 V at 10 mA until the dye front is 0.5 cm higher than that of the gel sealing.
7. Take the gel out between the plates by putting it into the plastic tray that contains distillate water. The gel should be cleaned for 1 minute. Then, rinse the gel with water and go to destain the stain procedure.

Staining and Destaining of Gel

1. After you have removed the water, add 50ml of Staining Solution inside the tray that contains gel until the bands appear apparent. Sometimes, the gel needs to be kept for a night inside the staining solution in order to ensure visualisation of bands.
2. Take the gel out of the Staining Solution. It is possible to reuse the Staining Solution can be re-used three times.
3. Rinse the gel with distilled water until the stain is removed of the gel. Repeat the process of changing the distilled water every 3-4 times.
4. Add 50ml of Destaining solution into the gel. Destaining should be performed by shaking the gel at a constant rate.
5. Continue to destain until the bands are clear and distinct. can be seen.
6. Take out the gel from the Destaining Solution. The Destaining Solution is able to be used three times.

Observation and Result of SDS-PAGE

• Lane 1: Prestained Protein Ladder
• Lane 3: Protein Sample 1
• Lane 5: Protein Sample 2 

Interpretation

Following staining, and then destaining, gel, you can compare the molecular mass of the samples to the weight of the protein marker. Protein Sample 1 is a serum sample so multiple bands are observed. Protein Sample 2 is a purified protein and therefore 1 major band is visible.

Sample Preparation for SDS-PAGE

1. Take 1ml of culture solution of E.coli strain DH5α, in 1.5 ml of eppendorf’s tube.
2. Then centrifuge at 12000 rpm for 5 min.
3. Then discard the supernatant.
4. Then add 500 μl of TE in the tube and dissolve the tube by gentle shaking.
5. Then add same volume of sample buffer.
6. Finally heat the sample on boiling bath for 5 minutes and then immediately keep on
   ice.

The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for
proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non–
disulphide linked tertiary structures, and additionally applies a negative charge to each protein in
proportion to its mass. Urea breaks the hydrogen bonds between the base pairs of the nucleic acid,
causing the constituent strands to separate. Heating the samples to at least 60 °C further promotes denaturation.

Important Instructions for SDS PAGE

• Take the time to read the entire process prior to beginning the test.
• Preparation of 10 percent APS Solution: Prior to beginning the experiment you need to dissolve 0.2 grams of Ammonium persulphate in water distillate to create an amount of 2.0 milliliters. Store at 2-8oC. Utilize within 3 months.
• Preparation of 1XTris-Glycine SDS Gel Run Buffer To make 500 ml 1X Tris-Glycine Gel running buffer, you need to take 100 ml of Tris-Glycine-SDS Gel Running Buffer and mix 400 ml of sterile distilled water*. Store at 2-8oC. Mix thoroughly prior to use. Make sure to mix well prior to use. 1X Tris-Glycine SDS Gel Running Buffer can be reused up to 4-5 times.
• Thaw all refrigerated samples prior to use.
• Cleanse the entire machine by using detergent and distillate water*. Check you have plates clean of dirt.

Problems with solution

• In low current, the Run takes an unusually long time, to solve this problem Increase the voltage by 25-50% 
• A Poor resolution of gel can occurs if Too much sample is loaded onto the wells, to avoid this Never overfill the wells as it may lead to artifacts. The given volume of gel loading is for a standard gel size. If gel size smaller load samples accordingly 
• Always pour stacking gel such that the length is 1 cm from the well bottom to the top of separating gel for proper stacking of protein sample 
• If Run taken place very fast, then Decrease the voltage by 25-50%, as current applied is very hig.
• Carry out the electrophoresis as soon as the sample is prepared if More bands seen for the purified protein sample.
• Allow the stacking gel to polymerize for atleast 30 minutes before removing the combs otherwise the Samples do not sink to the bottom of the well while loading.
• Make sure no bubbles are present in the gel when pouring (Bands on part of the slab do not move down the gel).
• Do not overtighten the screws on the clamp assembly.
• Ensure that the gel mixture is well mixed before pouring the gel