In SDS-PAGE, proteins are separated solely based on polypeptide chain length eliminating the influence of the structure and charge.
This course covers SDS-PAGE, principle involved, process and applications of SDS-PAGE.
Different proteins with similar molecular weights migrates differently due to their differences in secondary, tertiary or quaternary structure.
SDS, an anionic detergent, is used in SDS-PAGE to reduce proteins to their primary (linearized) structure and coat them with uniform negative charges, hence leading to their separation.
SDS ((or sodium lauryl sulphate) is detergent that binds non-covalently to proteins (Figure 1).
In the presence of SDS, the intrinsic charges of the protein molecules are masked.
During SDS-PAGE, all proteins migrate toward the anode (the positively charged electrode). As SDS-treated proteins have very similar charge-to-mass ratios, and similar shapes.
During the process, the rate of migration of SDS-treated proteins is effectively determined by their molecular weight.
A typical protocol of SDS-PAGE used for protein is as follows:
- Polyacrylamide gel (potential tissue embedding agent).
- The stacking gel, which is a large pore PAG (4%T). This gel is prepared with Tris/HCL buffer pH 8
- Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S; mW: 288.38)
- Ammonium persulfate
- Bromophenol Blue, Glycerol, Coomasie Brilliant Blue, n-butanol, Dithiothritol etc.
- The buffers
SDS-PAGE involves following steps (Figure 2):
Preparation of Tissue
Solution of proteins to be analyzed is mixed with SDS that denatures secondary and tertiary structures and applies negative charge to each protein in proportion to its mass.
Heating samples to at least 60° shakes the molecules, which allows SDS to bind with them.
A tracking dye is be added to the protein solution to track the progress of the protein solution through the gel during the electrophoretic run.
Preparation of Gel
The gel generally consists of acrylamide, bisacrylamide, SDS, and a Tris- HCl buffer with adjusted pH.
The ammonium persulfate and the TEMED are added when the gel is ready to be polymerized after degassing.
The separating or resolving gel is usually more basic and has higher polyacrylamide content than the loading gel. Add a thin layer of isopropanol.
Pour the loading gel and placed a comb to create the wells. After the loading gel is polymerized removed the comb and now the gel is ready for electrophoresis.
Preparation of Anode and Cathode Buffers
The anode buffer consists of Tris-Cl, distilled deionized water and is adjusted to a higher pH than the cathode buffer.
The cathode buffer contains SDS, Tris, Tricine, and distilled deionized water.
Setting the Electrophoretic Apparatus
Set the electrophoresis apparatus (Figure 3).
Load the protein sample on the slot by syringe or pipette (Figure 4).
Finally, hook the apparatus to a power source under appropriate running conditions to separate the protein bands.
Staining the Gel
Stain the gel. Most commonly used stains are Coomassie and Brilliant Blue R-250 or silver stain that allows visualization of the separated proteins.
Observe the bands obtained in the gel (Figure 6).
Applications of SDS-PAGE
SDS-PAGE is the most common method for separating proteins using polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) as detergent to denature the proteins.
- This techniques is widely used in forensics, genetics, molecular biology and biochemical research.
- Protein separation by SDS-PAGE is used to estimate relative molecular mass.
- It is used to determine the distribution of proteins among fractions, and relative abundance of major proteins in a sample.
- Specialized techniques, for example, Western blotting, 2-D electrophoresis, and peptide mapping uses SDS-PAGE. It helps in detection of extremely rare gene products, similarities among them, and for the separation of isoenzymes.
- Tricine-SDS PAGE is used for the separation of proteins in a range of 1 to 100 kDa.
- Other applications of SDS-PAGE include an evaluation of genetic diversity and purity, and differentiating cell wall proteins of different organisms.