The principle and Procedure of Polyacrylamide Gel Electrophoresis (SDS-PAGE)

sds page in dna extraction

SDS-PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) is a technique used to separate the proteins according to their masses.

 Separation of macromolecules under the influence of the charge is called electrophoresis. The gel used in SDA-PAGE is polyacrylamide and agent which is used to linearize the proteins is SDS. Hence the name SDS-PAGE.

Principle of SDS-PAGE:

Protein samples and ladder are loaded into wells in the gel and electric voltage is applied. A reducing agent such as mercaptoethanol or  dithiothreitol (DTT) (in the presence of a detergent i.e. SDS) breaks down the disulfide bridges that are responsible for protein folding; and a detergent such as SDS imparts negative charge to the proteins thereby linearizing them into polypeptides. Polyacrylamide provides a matrix for the polypeptides to run. Polypeptides run towards the positive electrode (anode) through the gel when electric field is applied. Electrophoretic mobility of the proteins depends upon 3 factors:

  • Shape – All the proteins are in the primary structure after the treatment with a reducing agent. So, shape doesn’t affect the protein separation.
  • Charge – All the proteins are negatively charge proportional to their molecular weight after treatment with SDS. So charge doesn’t affect the separation.
  • Size– proteins get separated solely on the basis of their molecular weight.

 Smaller polypeptides move faster because they have to face less hindrance, larger ones move slower because of greater hindrance. Hence proteins get separated ONLY on the basis of their mass (Fig. 01).

Principle of SDS-PAGE
fig. 01: Principle of SDS-PAGE

Functions of agents Used in SDS-PAGE

β-Mercaptoethanol /dithiothreitol (DTT)

It is mixed in the sample to reduce (break down) the disulfide bridges (S-S Bridges) present between the polypeptides of a protein between cysteine residues.. S-S bridges are responsible for the secondary structure of the proteins. Because electrophoretic mobility also depends upon the shape of the molecule. So it is very essential for all the protein molecules to be in the same shape. And this is achieved by using any reducing agent such as β-Mercaptoethanol. So, after the reduction the S-S bonds become –SH , -SH bonds leaving the peptide free. After the treatment with β-Mercaptoethanol all the proteins have primary structure hence only separated on the basis of size. (Fig. 01)

Sodium dodecyl sulfate (SDS)

SDS is mixed in sample buffer while preparing the sample for SDS-PAGE. After the treatment with β-Mercaptoethanol proteins are in one shape and after the treatment with SDS all the proteins get a net negative charge. SDS imparts negative charge to the proteins molecules evenly (Fig. 01). So, when the voltage is applies, all the proteins migrate towards the positive electrode of the gel. Because, proteins of different size get the negative charge proportional to their molecular weight, they only get separated by the size and not on the basis of the charge or shape. SDS is also present in the gel making sure that all the proteins should stay negative throughout the gel.

Bromophenol blue (BPB)

Bromophenol blue works as a tracking dye in electrophoresis and is used to monitor the progress of the molecules moving through the gel. It is mixed with the sample proteins. When the voltage is applied, BPB runs along with the proteins but faster. It reaches the end of the gel before any protein in the sample reaches it. Even the glycine molecules present in the running buffer reach after the BPB. BPB has a slightly negative charge that is why it moves towards the anode indicating the migration of the protein molecules.

Polyacrylamide gel

Polyacrylamide gel is manufactured by the polymerization of the monomer acrylamide in water by using small amount of a cross-linker e.g. N,N’-Methylenebisacrylamide. Hence both acrylamide and bisacrylamide copolymerize and makes a 3D network of straight chain of acrylamide with interconnection of bisacryamide (Fig 02).

acrylamide  interconnection  with  bisacryamide

Fig. 02: 3D network of straight chain of acrylamide with interconnection of bisacryamide

There are other cross-linkers available that are used instead of bisacrylamide. E.g.

  • piperazine diacrylate (PDA) – This  is used to reduce silver stain backgrounds in SDS-PAGE gels.
  • N,N’-diallyltartardiamide (DATD) –  It is a disruptable cross-linkers which enable gels to be solubilized.

The ratio of the acrylamide and bisacrylamide determines the pore size of the gel. So, in a discontinuous gel system different concentration and pH are used for stacking gel and resolving gel.

How the stacking gel works?

The purpose of the stacking gel is to stack the mixture of proteins that needs to be separated on the interface of stacking and resolving gel. Hence, all the proteins first get stacked on the line, and then run into the separating gel approximately on the same time no matter what is the size of the protein is.

Two types of molecules are responsible for this:

  1. trailing Glycine molecules from Tris-Gly electrophoresis buffer
  2. leading Chloride ions from Tris-HCL running buffer

When voltage is applied, glycine molecules (simplest amino acid) and Cl- begin to move through the gel towards the positive electrode. Since the glycine molecules are in the form of zwitter ions in stacking gel, their electrophoretic mobility is very low. Cl- ions move faster than the glycine molecules.an area of unbalanced positive counter ions is developed, thereby, developing a steep voltage gradient between chloride ions and glycine ions. Between the glycine molecules (slowest ones) and chloride ions (the fastest) exist all the proteins from the mixture.(Fig 03) Being intermediate in their mobility between chloride and glycine, the sample molecules are carried along becoming “stacked” into very thin, distinct layers in order of electrophoretic mobility.

At this stage, the protein molecules are trapped between the two ion fronts i.e. trailing glycine and leading Cl- .

A thin layer has been achieved at the boundary of stacking and resolving at the end of this stage.

stacking gel function

Fig. 03: Working of Stacking Gel

What happens on reaching interface between stacking gel and resolving gel?

On reaching the resolving gel, pH increases and pore size decreases abruptly. At much higher pH glycine molecules are no more in the form of zwitter ion, they become ionized at this stage and start to move faster than in stacking gel. Cl- ions travels towards the electrode in no time. So, Unstacking occurs, glycine molecules quickly overtake the sample protein molecules, leaving the protein molecules all free for the separation on the basis of their mass to charge ratio. (Fig. 04)

resolving gel

Fig. 04
  Stacking Gel Resolving gel
Polyacrylamide concentration Low High
Pore size Larger Smaller
pH of Tris-Cl used 6.8 8.8
Purpose To stack the polypeptides on the interface of stacking gel and resolving gel. To separate the polypeptides solely on the basis of size.
Electrophoretic mobility glycine <protein mixture <BPB <Cl. Protein mixture <glycine <BPB <Cl.

Ammonium persulfate and TEMED

Polymerization of the gel occurs by free radical mechanism. TEMED ((N,N,N,N-tetramethylethylenediamine) acts as a catalyst and generate free radicals of Sulfate. These sulfates for free radical generation is provided by ammonium persulfate as shown in the figure 02.

Major steps of SDS-PAGE

Pouring of the resolving gel:

Resolving gel is poured between two glass plates (one is called short plate and the other one is tall plate), clipped together on a casting frame (Fig. 05) Bubbles are removed by adding a layer of isopropanol on the top of the gel. (The level of the gel is predetermined by placing the comb on the glass-plates and leaving approximates 1cm space below the comb. Use a pen to mark the level. Now pour the gel up to this mark. ) The gel is then allowed to solidify. When the gel is solidified, remove the isopropanol by using a filter paper.

sds page casting frame
Fig. 05: Casting frame of SDS PAGE

Pouring of the stacking gel:

When the resolving gel is solidified, stacking gel is loaded all the way to the top of the glass plates. Comb is placed just after loading. The gel is, then, allowed to polymerize (solidify). When stacking gel is solidified, comb is removed very carefully not damaging the well’s shape.

Loading the ladder in wells

Add the ladder very carefully into the well which is on the extreme right using a micropipette. The samples are loaded into the other wells. Ladder is mostly pre-stained with the known molecular weight proteins. (Fig 06)

Loading the ladder in wells
Fig. 06: Loading the ladder in wells

Loading the samples in wells

Samples are loaded in each well with equal amount of the proteins mixture using micropipette. Be careful while loading the samples. Make sure not to damage the size of the wells or not to pour the sample out of the well instead of pouring inside it. At this stage, sample of the proteins appears to be blue because of a dye (bromophenol) used while preparing the sample.

Running the gel by applying voltage

A voltage is applied after dipping the “sandwich of gel and glass plates” in running buffer. Turn of the voltage when the tracking dye has reached or crossed the gel. The gel is further proceeded for the subsequent analysis.

Subsequent analysis – Coomassie Blue Staining

The gel is rinsed with deionized water 3-5 times to remove SDS and buffer. It may create hindrance with the binding of the dye (0.1% Coomassie Blue) to the proteins. The gel is then dipped in Coomassie Blue stain (staining buffer) on a shaking incubator at room temperature. The invisible bands of the proteins beginning to appear within minutes but it takes approximately 1h for complete staining (Fig 06).

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