In the previous posts, 2-Dimensional PAGE, Sample Preparation In 2-D PAGE, IPG strip Rehydration and Isoelectric Focusing (First Dimension), we have been discussing about the 2D-PAGE. After IEF, the second-dimension separation by SDS-PAGE has to be performed, which is done using either a flatbed or a vertical system, depending on various factors.
Now, the SDS-PAGE consists of four steps:
- Preparation of the second-dimension gel
- Equilibration of the IPG strip(s) in SDS buffer
- Placing the equilibrated IPG strip on the SDS gel
The equilibration step is common to both vertical and flatbed systems. The gel preparation, IPG strip placement, and electrophoresis protocols are specific to the orientation of the gel.
The second-dimension gel must be prepared before the equilibration step is started.
Equilibration: It is necessary to equilibrate the IPG strips in SDS-containing buffers before running the second dimension. It is a 2-step process which ensures that cysteines are reduced and alkylated to minimize or eliminate vertical streaking. Equilibration Buffer I contain DTT, while Equilibration Buffer II contains iodoacetamide.
one of the commonly used composition of the Buffer I and II are
Buffer I (With DTT): 6 M urea, 0.375 M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol, 2% (w/v) DTT
Buffer II (With Iodoacetamide): 6 M urea, 0.375 M Tris-HCl, pH 8.8, 2% SDS, 20% glycerol, 2.5% (w/v) iodoacetamide
Equilibration introduces reagents essential for the second-dimension separation. Equilibration buffer maintains IPG strip pH in an appropriate range for electrophoresis. Electroendosmosis may occur due to the presence of fixed charges on the IPG strip in the electric field and can interfere with protein transfer from the IPG strip to the second-dimension gel. Glycerol (30%) together with urea reduces electroendosmosis and improves transfer of protein from the first to the second-dimension. DTT preserves the fully reduced state of denatured, unalkylated proteins. Sodium dodecyl sulfate (SDS) denatures proteins and forms negatively charged protein-SDS complexes. The amount of SDS bound to a protein, and therefore the additional negative charge, is directly proportional to the mass of the protein. Thus, electrophoresis of proteins through a sieving gel in the presence of SDS separates proteins on the basis of molecular mass. Iodoacetamide alkylates thiol groups on proteins, preventing their reoxidation during electrophoresis. Protein reoxidation during electrophoresis can result in streaking and other artifacts. Iodoacetamide also alkylates residual DTT to prevent point streaking and other silver-staining artifacts. Iodoacetamide is introduced in a second equilibration step. This step is optional when SDS-PAGE is performed in a vertical second-dimension system, but required when SDS-PAGE is performed on a flatbed second-dimension system especially when the flatbed separation is to be visualized by silver staining. The second equilibration with iodoacetamide is also used to minimize unwanted reactions of cysteine residues (i.e. when mass spectrometry is to be performed on the separated proteins). Tracking dye (bromophenol blue) allows monitoring of electrophoresis
The gel for the SDS PAGE is prepared before the equilibration. After equilibration, the IPG strip is dipped briefly into 1x Tris- glycine-SDS running buffer.
Then the agarose solution is overlaid into the IPG well of the gel. IPG strip is then carefully pushed into the well taking care to avoid air bubbles beneath the IPG strip. Alternatively, the IPG strips can be pushed in contact with the second-dimension gel surface prior to applying the agarose solution over the IPG strip (this alternative method is often necessary with second-dimension gels ≥ 20 cm in width, as the agarose solidifies before the IPG strip can be properly inserted).
Once the agarose solidifies the gel is mounted and electrophoresis is carried out. The migration of the Bromophenol Blue, present in the overlay agarose solution, is used to monitor the progress of the electrophoresis
This is a technique of denaturing electrophoresis. SDS-PAGE (SDS-polyacrylamide gel electrophoresis) is an electrophoretic method for separating polypeptides according to their molecular weights (Mr). The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate (SDS). SDS is an anionic detergent. When in solution in water, SDS forms globular micelles composed of 70–80 molecules with the dodecyl hydrocarbon moiety in the core and the sulfate head groups in the hydrophilic shell. SDS and proteins form complexes with a necklace-like structure composed of protein-decorated micelles connected by short flexible polypeptide segments. The result of the necklace structure is that large amounts of SDS are incorporated in the SDS-protein complex in a ratio of approximately 1.4 g SDS/g protein. SDS masks the charge of the proteins themselves and the formed anionic complexes have a roughly constant net negative charge per unit mass. Thus, electrophoresis of proteins through a sieving gel in the presence of SDS separates proteins on the basis of molecular mass.
Besides SDS a reducing agent such as dithiothreitol (DTT) is also added to break any -S-S-linkages present in the proteins. When proteins are treated with both SDS and a reducing agent, the degree of electrophoretic separation within a polyacrylamide gel depends largely on the molecular weight of the protein. In fact, there is an approximately linear relationship between the logarithm of the molecular weight and the relative distance of migration of the SDS-polypeptide complex.
The most commonly used buffer system for second-dimension SDS-PAGE is the tris-glycine system described by Laemmli. This buffer system separates proteins at high pH, which confers the advantage of minimal protein aggregation and clean separation even at relatively heavy protein loads. The Laemmli buffer system has the disadvantage of a limited gel shelflife.
Thus, at the end of second dimension, the proteins are separated according to their pI in the first dimention and further the separated proteins are separated according to their molecular mass in the second dimension at the perpendicular directional of the first dimension. Hence the separation is far better than obtained through a single electrophoresis technique. Hence this is a great method to separate a very complex mixture of proteins.
In the next blog I will discuss about the visualization of the proteins in the gel.