SDS PAGE technique

SDS PAGE or sodium dodecyl sulphate polyacrylamide gel electrophoresis  is a gel separation technique

commonly used to separate proteins. This technique was developed by Ulrich K Laemmli  and is a discontinuous electrophoretic gel separation method. Due to the combined effect of SDS and poly acrylamide the proteins are separated on the basis of their molecular weight.

SDS and acrylamide are the two fundamental components of this technique .SDS is an anionic detergent that acts as a surfactant and helps in denaturing the proteins and their migration based on their molecular weights. SDS confers negative charges to the proteins, masking the proteins intrinsic charge and providing them a charge according to their mass. In SDS concentrations above 0.1 mM most protein denature. Certain proteins that contain s-s linkage may resist denaturation and in these cases an additional method , heating is used.

β mercaptoethanol in the sample buffer too helps in breaking the S-S linkages.

SDS PAGE

• Gel preparation
• Sample preparation
• Electrophoresis
• Protein Staining\Western blotting

GEL PREPARATION

SDSPAGE  involves two gel set one on top of other. The top gel is the stacking gel and the bottom gel is the resolving or separating gel. Both the gel have the same components , they only differ in the pH of their tris buffers which in turn decides whether the gel act as a stacking gel(pH6.8) or a resolving gel( pH8.8).Proteins are highly concentrated in the stacking gel prior to their migration to the separating gel. Acrylamide is used as a gel former. Different concentrations of acrylamide is used to separate proteins of different molecular weights. For instance , a high percentage of acrylamide gel is used in case of smaller proteins. Higher the acrylamide concentration smaller the pore size in the gel. With regard to the type of sample protein you are resolving gels of concentrations 6%- 12% can be prepared.

The above table depicts the the various amounts of components needed to prepare different concentrations of the resolving gel. The basic components for both the gels are the same, Water,1.5 M Tris, 30%acrylamide, 10%SDS 10% APS( Ammmonium persulpahte) and TEMED(Tetramethylethylenediamine ).

APS acts as the initiator for the acrylamide polymerization and TEMED as an catalyst .

Gels are set in sealed glass plates with spacers in between them. First the separating gel is poured upto 3\4th mark of the glass plate , followed by stacking gel (after an interval of 30 minutes) in which the comb is placed.

While preparing the gel solution, TEMED and APS are added at the last , as the polymerization  starts immediately after these two are added to the solution. Therefore the gel solution once mixed is immediately poured into the casting setup. Resolving gel requires about 30-40 minutes to set . Once the resolving gel is poured between the glass plates, a layer of water is poured above the gel to cut off the oxygen, to ensure better polymerization.

.Once the resolving gel is set ,  water on top of the gel is discarded. Next, the mixture for stacking gel is prepared and poured on top of the resolving gel. Comb is inserted in between the glass plates and into the stacking gel. The combs are removed after about 10-20 minutes and we end up with well designed wells for our samples to be loaded The well are washed with distilled water to remove and unpolymerized acrylamide and gel pices .This brings us to the next step of our protocol

SAMPLE PREPARATION

Protein samples are first mixed with sample buffer(SDS gel loading buffer) and then denatured by keeping in water bath at 100° C for 3 minutes. The sample is then centrifuged at 15000 rpm for 1 minute at 4C and the collected supernatant is used for SDS PAGE.

SDS gel-loading buffer (2X)

100 mM Tris-Cl (pH 6.8)

4% (w/v) SDS (sodium dodecyl sulfate; electrophoresis grade)

0.2% (w/v) bromophenol blue

20% (v/v) glycerol

200 mM DTT (dithiothreitol)

Store the SDS gel-loading buffer without DTT at room temperature. Add DTT from a 1 M stock just before the buffer is used.

200 mM β-mercaptoethanol can be used instead of DTT.

Bromophenol blue in the gel loading buffer gives the sample a blue color facilitating visualization of the sample. while electrophoresis. Whereas SDS is used as a denaturing agent. DTT or β-mercaptoethanol is used as a reducing agent to further denature the proteins that resist SDS or have S-S linkages. These allow better separation during electrophoresis. Glycerol is much more dense than water and is added to make the sample fall to the bottom of the sample well rather than just flow out and mix with all the buffer in the upper reservoir.

ELECTROPHORESIS

SDS-PAGE 10× SDS Running Buffer

Once the gels are set, the spacers are removed and the glass plate with the gel is placed into the electrophoretic chamber. The cathode and anode are plugged , running buffer is poured into the SDS PAGE apparatus. The prepared protein samples are then  loaded into each well and electrophoresis is run. The gel is run until the dye bromophenol blue in the sample buffer reaches the bottom of the gel.

PROTEIN STAINING

After the gel is run it is separated from it is gently using a spatula separated from the glass plates and transferred in the staining solution. The gel gel kept in the staining solution overnight to ensure that the stain Coomassie blue binds with the protein bands in the gel. Later, the gel after a rinse in DDW is transferred to the destaining solution which distains and clears the gel of all stain except at the protein bands.

This completes the basic procedure of SDS PAGE .

Role of Glycine

Now let’s discuss the role of glycine in SDS PAGE. As mentioned earlier in the stacking gel all the protein stack on one another and as they hit the resolving gel , they start separating based on their molecular sizes.

So how do the proteins end up stacked. This is where glycine performs it’s role.

Glycine is an amino acid with the chemical formula NH₂-CH₂-COOH. The charge of its ion is dependent on the pH of the solution that it is in. In acidic environments,  glycine molecules become positively charged. At a neutral pH of around 7, the ion is uncharged (a zwitterion), having both a positive charge and a negative charge and at a higher pH, glycine becomes more negatively charged.

So how does glycine help in stacking of the proteins?
Glycine is in the running buffer, which is typically at a pH of 8.3. At this pH, glycine is predominately negatively charged, forming glycinate anions. When an electric field is applied, glycinate anions hit the pH 6.8 stacking buffer, and change to become mostly neutrally charged glycine zwitterions. As zwitterions these glycine molecules moves towards the anode at a very slow speed because they carry no charge.

By contrast, the Cl- ions (from the Tris-HCl in the gel) move at a faster rate towards the anode. When the Cl- and glycine zwitterions hit the loading wells with the protein samples, they create a narrow but steep voltage gradient in between the highly mobile Cl- ion front (leading ions) and the slower moving, more neutral glycine zwitterion front (trailing ions). The mobility of the protein sample lies in between the highly mobile Cl and the slow moving glycine zwitter ion, making the proteins herded between them.

When they hit the resolving gel the glycine ions at a sudden stage acquire a lot of negative charge, thereby taking off towards the positively charged anode at a higher pace. So now these glycine anions speed past the proteins towards the anode leaving proteins stacked at the top of the resolving gel.

When in the resolving gel the proteins slowly separate according to their molecular weights.