What is SDS-PAGE electrophoresis?
SDS-PAGE electrophoresis, short for sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a kind of commonly used protein extraction technology to polyacrylamide gel is made up of acrylamide (Acr) and cross-linking agent N, N ‘a methylene double acrylamide (Bis) in catalyst ammonium persulfate (APS), N, N, N’, N ‘tetramethylethylenediamine (TEMED) was used as a support for electrophoresis. It also called vertical electrophoresis, and because it is qualitative detection of proteins, also called protein electrophoresis.
Principles of SDS-PAGE
SDS is an anionic detergent. When SDS and mercaptoethanol are added to protein sample solution, mercaptoethanol can open disulfide bonds, and SDS can open non-covalent bonds (hydrogen bonds, hydrophobic bonds, ionic bonds) of proteins, and bind to protein molecules. Because SDS carries a large amount of negative charge, SDS-protein complex carries a large amount of negative charge, which greatly exceeds the original charge amount of protein molecules, thus masking the original charge difference between different types of proteins.
1. The reason why mercaptoethanol opens the disulfide bond: the sulfhydryl reaction of the cysteine side chain is very high, and dissociation can occur at a slight base, while the disulfide bond oxidant reducing agent can be opened, mercaptoethanol can reduce the disulfide bond to 2R-SH, and a stable structure, so that the balance moves to the right.
2. Why open the disulfide bond? Because disulfide bonds fold proteins and interfere with electrophoretic migration, it is important to eliminate the effect of protein conformation. Disulfide bonds and non-covalent bonds open, protein conformation changes, and the mobility of the protein-SDS complex in the gel is not affected by the original charge and shape of the protein, but only by the relative molecular weight of the protein, so different protein samples can be electrophoretic on the same piece of protein glue.
Three factors of protein electrophoresis mobility
Shape: All proteins are in a primary structure after treatment with reducing agents. Therefore, the shape does not affect the separation of proteins.
Charge: After SDS treatment, all proteins are negatively charged, so the charge does not affect the separation.
Size: The separation of proteins is completely related to their molecular weight size.
In electrophoresis, peptides with smaller molecular weights move faster because they encounter less resistance. Peptides with larger molecular weights move more slowly because they encounter more resistance. So proteins are separated entirely by their molecular weight.
Main reagents for SDS-PAGE
10% sodium dodecyl sulfate (SDS): anion stain remover, which causes the protein to carry a large amount of negative charge, dissociates the hydrogen bond between the proteins, cancelates the hydrophobic action within the protein molecules, and removes polypeptide folding.
30% acrylamide (Bis-Acr): provides the carrier, and its solidification is directly related to the success of electrophoresis. The straight chain of acrylamide and the interconnection of double acrylamide form a three-dimensional network structure. Acrylamide is highly neurotoxic and can be absorbed through the skin or into the respiratory tract. Wear a mask and gloves when doing experiments.
10% ammonium persulfate (APS): Ammonium persulfate can be hydrolyzed into ammonium bisulphate and hydrogen peroxide in aqueous solution, providing free radicals for the preparation of PAGE glue. Ammonium persulfate is easy to deliquesce failure, it is recommended to separate small bottles. After opening, it is recommended to prepare ammonium persulfate into 10% aqueous solution at one time and freeze it at -20 degrees.
TEMED: A polymer formed by acrylamide polymerization, its function is that the polymerization reaction is free radical polymerization, and an initiator is required to generate free radicals and trigger the reaction to induce ammonium persulfate, which is the initiator, and TEMED can catalyze ammonium persulfate to produce free radicals, thereby accelerating the polymerization of acrylamide gel. TEMED has a strong pungent smell, pay attention to protection when using.
Bromophenol blue (BPB): Bromophenol blue is a tracer dye used in electrophoresis to monitor the progress of the electrophoresis process of individual protein molecules. When electrophoresis begins, the BPB migrates with the protein, but faster, reaching the end of the gel faster than any protein in the sample, and even the glycine molecules in the buffer reach the end after the BPB. BPB has a slight negative charge, which is why it can migrate towards the anode and at the same time act as a tracer dye for protein molecules.
SDS-PAGE gel electrophoresis protocol
Materials and reagents required for SDS-PAGE
Vertical electrophoresis room with power supply, glass plate, gasket and comb.
| Chemical name | CAS | Concentration/ratio/amount | Purpose description |
| Acrylamide | 79-06-1 | Typically a 30% acrylamide solution for preparing separating and stacking gels | Main component for forming the gel |
| Bis-acrylamide | 110-26-9 | Usually mixed with acrylamide at a ratio of 29:1 | Crosslinking agent, forms a three-dimensional network with acrylamide |
| SDS (sodium dodecyl sulfate) | 151-21-3 | Typically used at a concentration of about 0.1% in gels and sample buffer | Causes proteins to carry a negative charge for electrophoretic separation |
| Instant premixed granules of Tris-HCl | / | Separating gel usually uses 1.5M Tris-HCl pH 8.8, and stacking gel uses 6M Tris-HCl pH 6.8 | Maintains pH stability |
| TEMED (tetramethylethylenediamine) | 110-18-9 | Added in small amounts to the gel solution | Catalyst, accelerates the polymerization reaction |
| APS (ammonium persulfate) | 7727-54-0 | Added in small amounts to the gel solution | Initiator, starts the polymerization reaction |
| Bromophenol blue | 115-39-9 | Usually mixed with samples and loaded onto the gel together | Sample tracking dye, used to observe the migration of samples in the gel |
| Sample buffer | 60-24-2 | β-mercaptoethanol | Contains SDS, glycerol, bromophenol blue, etc., for sample preparation |
| 56-81-5 | Glycerol | ||
| 3483-12-3 | Dithiothreitol | ||
| Gel staining solution | 6104-58-1 | Coomassie brilliant blue | For staining the gel to visualize proteins in the gel |
| / | silver staining reagents | ||
| Gel destaining solution | 67-56-1 | methanol | To remove unbound dye from the gel to improve staining effect |
| 64-19-7 | Acetic acid |
Glue preraration and filling
Glue preparation: strictly in accordance with the formula of glue, must be mixed.
If the mixture is not uniform, it may cause the glue not to solidify, or the sample is skewed; In the past, the separation glue was first made and then the concentrated glue, but now there is a ready-made kit, which can be prepared together with the glue.
Glue filling: The prepared lower layer of glue is injected between two glass plates, and the height can be positioned with a comb, about 1cm below the comb. Shake gently to make the page level, seal the glue with water, pour away the water after the lower glue has solidified, continue to prepare the upper glue, again, mix well, fill the glue, insert the comb, after the glue has solidified, set aside for use.
The finished glue can also be stored at 4 ℃, remember to add a little electrophoresis buffer, so as not to dry the glue, no problem for a week.
Protein sample preparation
The sample to be tested is treated with sample buffer, sample: sample buffer = 8:2 or 9:1, after mixing, boiling water bath for 5-10 min.
If it’s an intracellular product, remember to break up the cell, like a protein expressed by E. coli. In the saturated state, the combination of SDS and protein is proportional to the mass (i.e. 1.4g SDS /g protein), and the protein content can not exceed the standard, otherwise the SDS binding amount is insufficient.
Sample loading
The electrophoresis tank was filled with electrophoresis buffer before passing through the sampling hole, and the whole device was tilted to remove any bubbles that might remain under the gel. The sampling was added in the predetermined sequence, with 20 μL added to each hole and 5 μL protein marker added to the protein Marker hole.
Electrophoresis
Cover the upper cover and wire, set the voltage and current to start electrophoresis, and stop electrophoresis when bromophenol blue reaches the bottom of the electrophoresis tank for about 1 h.
Generally, the concentrated glue voltage is 80-100 V, and the separation glue voltage is high, 120-150 V. If the result is anxious, the voltage can be increased appropriately.
Gel cutting and staining
Remove the rubber plate, cut the rubber plate, and gently put it into the dyeing tank for about 20-30 min. Appropriate heating, can add color dyeing.
It’s already dyed with bromophenol blue. Why dye it? After loading, the molecular weight of bromophenol blue trotted fast, the molecular weight of protein ran slowly, and gradually separated, and the protein had no color, and the protein bands could not be observed without staining. If it is recycled dye, extend the dyeing time appropriately.
Destaining
After dyeing, recover the dyeing solution, rinse the gel with pure water 2-3 times, and then put it in the decoloring tank, add the decoloring solution for decoloring until there is no background color. You can put a piece of absorbent paper on the side of the decolorization tank for cleaner decolorization. Can be properly heated after decolorization to reduce the decolorization time.
Gel imaging and analysis
The analytical protein glue can be compared by gray scale method or compared with standard protein.
Application of SDS-PAGE
Estimate the molecular weight of the protein.
Estimate the purity of the protein.
Used to analyze the size of polypeptide subunits and peptide map analysis, etc.
Other blogs
Magnetic Beads (MBs) for Protein Purification
People also like