Introduction
- SDS-PAGE is a technique used by many researchers to separate mixtures of proteins by their mass.
- Sodium dodecyl sulfate (SDS) is a detergent that breaks up the interactions between proteins.
- SDS is an anionic detergent that disrupts secondary and non-disulfide-linked tertiary structures and additionally applies a negative charge to each protein in proportion to its mass
- Polyacrylamide gels are formed through the copolymerization of acrylamide and bis-acrylamide (N,N’-methylenebisacrylamide), resulting in a highly cross-linked matrix. This gel functions as a molecular sieve, allowing for the separation of proteins based on their size under the influence of an electric field.
Components of SDS-PAGE
Polyacrylamide gel
- The gel used for SDS-PAGE is made out of acrylamide which form cross-linked polymers of polyacrylamide.
- Standard gels are typically composed of two layers, one top-most layer called the stacking gel and a lower layer called separating or resolving gel.
- The stacking layer contains a low percentage of acylamide and has low pH , while the acrylamide concentration of the separating gel varies according to the samples to be run and has higher pH.
- The difference in pH and acrylamide concentration at the stacking and separating gel provides better resolution and sharper bands in the separating gel.
- This gel material can also withstand high voltage gradients, is amenable to various staining and destaining procedures, and can be digested to extract separated fractions or dried for autoradiography and permanent recording.
Stacking and resolving gel
Stacking gel:
- The stacking gel is a large pore PAG (4%T). This gel is prepared with Tris/HCl buffer pH 6.8 of about 2.0 pH units lower than that of electrophoresis buffer (Tris/Glycine).
- This gel is cast over the resolving gel. The height of the stacking gel region is always maintained more than double the height and th volume of the sample to be applied.
Resolving gel:
- The resolving gel is a small pore polyacrylamide gel (3 -30% acrylamide monomer) typically made using a pH 8.8 Tris/HCl buffer.
- In the resolving gel, macromolecules separate according to their size. Resolving gels have an optimal range of separation that is based on the percent of monomer present in the polymerization reaction.
Components of SDS-PAGE:
The components of an SDS PAGE gel electrophoresis system are the following:
- A Slab holder for vertical or horizontal gels (thin, flat sheets of many individual lanes)
- Polyacrylamide or agarose gels (cm x cm x mm); these are poured for each analysis
- Gel is amended with SDS to dissociate & charge proteins.
- High voltage power supply (0.1-6 kV)
- A detection technique (dye staining, fluorescence, or autoradiography to image separated bands)
SDS-PAGE stands for Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis. Sodium- Dodecyl Sulfate, (“SDS”), is an anionic detergent. It is composed of a hydrophilic group with a net negative charge and a long hydrophobic chain with neutral charge.
Sample preparation
Samples may be any material containing proteins or nucleic acids. These may be biologically derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental samples, or purified proteins.
- In the case of solid tissues or cells, these are often first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or by using cycling of high pressure, and a combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – may be used to separate different cell compartments and organelles prior to electrophoresis. Synthetic biomolecules such as oligonucleotides may also be used as analytes.
- The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS for proteins or urea for nucleic acids.
- SDS denatures secondary and non–disulfide–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.
- In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta- mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE.
- A tracking dye may be added to the solution. This typically has a higher electrophoretic mobility than the analytes to allow the experimenter to track the progress of the solution through the gel during the electrophoretic run.
Preparation of ACRYLAMIDE GELS:
- The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent the formation of air bubbles during polymerization. Alternatively, butanol may be added to the resolving gel (for proteins) after it is poured, as butanol removes bubbles and makes the surface smooth.
- A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added to initiate polymerization. The polymerization reaction creates a gel because of the added bisacrylamide, which can form cross-links between two acrylamide molecules. The ratio of bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part in 35.
- The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight molecules, while much higher percentages are needed to resolve smaller proteins.
- Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis.
Electrophoresis
- Various buffer systems are used in PAGE depending on the nature of the sample and the experimental objective. The buffers used at the anode and cathode may be the same or different.
- The gel is run usually for a few hours, though this depends on the voltage applied across the gel; migration occurs more quickly at higher voltages, but these results are typically less accurate than at those at lower voltages. After the set amount of time, the biomolecules have migrated different distances based on their size.
- Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point of origin. Biomolecules may therefore be separated roughly according to size, which depends mainly on molecular weight under denaturing conditions, but also depends on higher-order conformation under native conditions. However, certain glycoproteins behave anomalously on SDS gels.
Components of SDS-PAGE:
Chemical buffer:
Stabilizes the pH value to the desired value within the gel itself and in the electrophoresis buffer. The buffer should also be unreactive and not modify or react with most proteins. Different buffers may be used as cathode and anode buffers, respectively, depending on the application. Multiple pH values may be used within a single gel, for example in DISC electrophoresis. Common buffers in PAGE include Tris, Bis-Tris, or imidazole.
Counter-ion:
Balance the intrinsic charge of the buffer ion and also affect the electric field strength during electrophoresis. Highly charged and mobile ions are often avoided in SDS- PAGE cathode buffers, but may be included in the gel itself, where it migrates ahead of the protein.
Acrylamide (C3H5NO):
When dissolved in water, slow, spontaneous autopolymerization of acrylamide takes place, joining molecules together by head on tail fashion to form long single- chain polymers. The presence of a free radical-generating system greatly accelerates polymerization. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires linking various chains together. Acrylamide is a neurotoxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.
Bisacrylamide (N,N′-Methylenebisacrylamide) (C7H10N2O2):
Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels. Chemically it can be thought of as two acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide can crosslink two polyacrylamide chains to one another, thereby resulting in a gel.
Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S):
SDS is a strong detergent agent used to denature native proteins to unfolded, individual polypeptides. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass-charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.
Urea (CO(NH2)2):
Urea is a chaotropic agent that increases the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds and van der Waals forces. Macromolecular structure is dependent on the net effect of these forces, therefore it follows that an increase in chaotropic solutes denatures macromolecules,
Ammonium persulfate (APS) (N2H8S2O8):
APS is a source of free radicals and is often used as an initiator for gel formation. An alternative source of free radicals is riboflavin, which generated free radicals in a photochemical reaction.
TEMED (N, N, N′, N′-tetramethylethylenediamine) (C6H16N2):
TEMED stabilizes free radicals and improves polymerization. The rate of polymerisation and the properties of the resulting gel depend on the concentrations of free radicals. Increasing the amount of free radicals results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount shows the reverse effect. The lowest catalytic concentrations that allow polymerisation in a reasonable period of time should be used. APS and TEMED are typically used at approximately equimolar concentrations in the range of 1 to 10 mM.
Chemicals used for processing and visualization
Tracking dye:
As proteins and nucleic acids are mostly colorless, their progress through the gel during electrophoresis cannot be easily followed. Anionic dyes of a known electrophoretic mobility are therefore usually included in the PAGE sample buffer. A very common tracking dye is Bromophenol blue (BPB, 3′,3″,5′,5″ tetrabromophenolsulfonphthalein). This dye is coloured at alkali and neutral pH and is a small negatively charged molecule that moves towards the anode. Being a highly mobile molecule it moves ahead of most proteins. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can weakly bind to some proteins and impart a blue colour. Other common tracking dyes are xylene cyanol, which has lower mobility, and Orange G, which has a higher mobility.
Loading aids:
Most PAGE systems are loaded from the top into wells within the gel. To ensure that the sample sinks to the bottom of the gel, sample buffer is supplemented with additives that increase the density of the sample. These additives should be non-ionic and non-reactive towards proteins to avoid interfering with electrophoresis. Common additives are glycerol and sucrose.
Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2):
CBB is the most popular protein stain. It is an anionic dye, which non-specifically binds to proteins. Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated into the gel can be removed by destaining with the same solution without the dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic, it may interfere with staining process. Therefore, large volume of staining solution is recommended, at least ten times the volume of the gel.
Ethidium bromide (EtBr):
It is the traditionally most popular nucleic acid stain.
Silver staining:
It is used when more sensitive method for detection is needed, as classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band, Silver staining increases the sensitivity typically 50 times. Silver staining was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels.
Western Blotting:
It is a process by which proteins separated in the acrylamide gel are electrophoretically transferred to a stable, manipulable membrane such as a nitrocellulose, nylon, or PVDF membrane. It is then possible to apply immunochemical techniques to visualise the transferred proteins, as well as accurately identify relative increases or decreases of the protein of interest.
Applications of SDS-PAGE.
- Measuring molecular weight.
- Peptide mapping.
- Estimation of protein size.
- Determination of protein subunits or aggregation structures.
- Estimation of protein purity.
- Protein quantitation.
- Monitoring protein integrity.
- Comparison of the polypeptide composition of different samples.
- Analysis of the number and size of polypeptide subunits.
- Post-electrophoresis applications, such as Western blotting.
- Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid.
- Pouring and Running a Protein Gel by reusing Commercial Cassettes.
- Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes.
- Detection of Protein Ubiquitination.
- SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval.
Advantages and disadvantages of SDS-PAGE
Advantages
- Migration of the molecules is proportional to their molecular weights.
- Highly sensitive test, separates molecules that have even a 2% difference in mass.
- Requires very small amounts of samples.
- A stable chemically cross-linked gel is used.
Disadvantages
- Poor band resolution due to high alkaline operating pH.
- Acrylamide gel is a potent neurotoxin chemical.
- Gel preparation is difficult and takes a long time.
- Very costly.
References
- Keller, Silke & Liedek, Anke & Shendi, Dalia & Bach, Monika & Tovar, Günter & Kluger, Petra & Southan, Alexander. (2020). Eclectic characterisation of chemically modified cell-derived matrices obtained by metabolic glycoengineering and re-assessment of commonly used methods. RSC Advances. 10. 35273-35286. 10.1039/d0ra06819e.
- Peffers, Mandy & Thorpe, Chavaunne & Collins, John & Eong, Robin & Koh, Timothy & Screen, Hazel & Clegg, Peter. (2014). Proteomic Analysis Reveals Age-related Changes in Tendon Matrix Composition, with Age- and Injury-specific Matrix Fragmentation. The Journal of biological chemistry. 289. 10.1074/jbc.M114.566554
- Bio-Rad Laboratories. (2023). Protein electrophoresis handbook. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf
- Bollag, D. M., Edelstein, P. A., & Swaney, P. B. (1996). Protein methods. John Wiley & Sons.
- Walker, J. M. (2000). The protein protocols handbook. Humana Press.