Protein electrophoresis is a protein analysis technique. Proteins are negatively or positively charged in a buffer and move to the anode in an electric field called electrophoresis. Different protein molecules have different electrophoretic mobilities. A very important part of protein electrophoresis is gel preparation. Conventional gel preparation often takes several hours, and there are also problems such as liquid leakage, uneven gel, and air bubbles. In addition to the trouble of preparation, the raw material reagents will also have certain toxicity, which will cause harm to the body of the glue dispenser, and the appearance of prefabricated glue can solve these troubles. So what is the basic principle of protein electrophoresis, and what is the function and principle of precast gel?
1. What is the basic principle of protein electrophoresis?
2. What is the function and principle of prefabricated glue?
3. Introduction of prefabricated glue provided by Yeasen
5. Product details
Most biological macromolecules, such as proteins and nucleic acids, have cationic and anionic groups, which are called zwitterions. The zone electrophoresis method in which the particles are dispersed in the solution and the starch gel, agar or agarose gel, polyacrylamide gel, etc. as the supporting medium is called gel electrophoresis. Polyacrylamide gel electrophoresis is an electrophoresis method in which the support is a polyacrylamide gel. It is believed that the size of the pore size of the polyacrylamide gel is controlled to polymerize, and the proteins are separated by the action of a similar molecular sieve.
Protein electrophoresis includes two forms, native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Native-PAGE keeps proteins intact during electrophoresis and separates them in a gradient based on their molecular weight, shape, and accompanying charge. On the other hand, SDS-PAGE separates proteins according to the molecular weight of their subunits. SDS is an anionic detergent. As a denaturant and a solubilizing agent, it can break intramolecular and intermolecular hydrogen bonds, fold molecules, and destroy the secondary and tertiary structures of protein molecules. As a strong reducing agent, mercaptoethanol, dithiothreitol, can break the disulfide bonds between cysteine residues. After adding the reducing agent and SDS to the sample and gel, the molecules are depolymerized into polypeptide chains, and the depolymerized amino acid side chains and SDS are combined into protein-SDS micelles. The negative charge of protein-SDS micelles exceeds the original charge of the protein, eliminating the charge difference and structural difference between different molecules. The mobility of the protein depends only on the molecular mass of the protein, and the molecular mass of the protein can be determined by comparing it with a standard of known molecular mass.
Compared with traditional handcast gels, precast protein gels can avoid contact with a large number of toxic reagents, save a lot of time, and effectively solve the problem of large differences in handcast gels.
The gel consists of two different gel layers, the upper layer is a stacking gel and the lower layer is a separating gel. Stacking gel is also known as stacking gel. The gel concentration is relatively small and the pore size is relatively large. The sample is added to the stacking gel and concentrated in a narrow zone through the migration of the large pore-size gel. Separating gel, also known as electrophoresis gel, usually has a small pore size, and selecting the appropriate gel concentration can make the sample components well separated.
Separating gels can be divided into fixed gels and gradient gels. The concentration of polyacrylamide is 8%, 10%, 12%, 15%，4% to 12%, and 4% to 20%. The first four are precast gels with a fixed concentration, and the last two are gradient gels. The concentrations represent the range of isolated protein molecular weights. Gel gradients are formed by gradient mixers. A high-concentration acrylamide solution is added to the glass plate, and then the concentration of the solution decreases in a gradient, and the pore size of the gel is larger at the top and smaller at the bottom. The fixative gel can only separate proteins within its separation range, neither too large nor too small. Gradient gels do not have this limitation, where large molecules are separated at the top and small molecules are separated at the bottom. Since the pore size of the gradient gel gradually becomes smaller, it has a concentrating effect on the protein near the pore size that the protein cannot penetrate, so a very narrow zone will be formed after electrophoresis. Proteins with small differences in molecular weight often cannot be separated on fixed gels but can be resolved on gradient gels.
YEASEN Precast Protein Plus Gel contains gradient concentration and fixed concentration, a variety of different concentrations can meet the needs of different protein electrophoresis. It is prepared by a unique gel preparation process and has an excellent electrophoretic effect. It is compatible with all kinds of electrophoresis tanks on the market (Bio-RAD, Tanon, Life, etc.) and has the advantages of wide adaptability, high resolution, high stability, clear and uniform bands, etc.
3.1 YEASEN Precast Protein Plus Gel upgrade to a plastic board, better quality!
Easy to use: ready to use; Tear off the adhesive tape from the bottom of the plastic board
Wide range of applications: compatible with denatured protein and natural protein
Clear band: the special treatment of plastic plates greatly reduces protein adsorption
Time-saving: the experiment can be completed in 18 minutes at the fastest
Quality assurance: strict inter-batch stability assurance
Long shelf life: can be stored at 4℃ for 12 months
3.2 The following parameters can help you choose the appropriate gel series
Electrophoresis buffer system: denatured protein running buffer or natural protein running buffer
Number of wells and sample loading: 10 or 15 wells, the maximum sample size was 70 μL and 40 μL, respectively
gel concentration: 8%, 10%, 12%, 15%, 4%-12%, 4%-20%
Table 1. Gel concentration and range of linear separation
|gel concentration||Range of linear separation|
3.3 Data show
Figure 1. Data show
Figure 2. Data show
Q1: Must the precast gel be placed at 4℃? Does it matter if you leave it at room temperature for a while?
A: Recommended 4℃, can be stored for 12 months. Do not store below 0℃, can be stored at room temperature (25℃) for 30 days.
Q2: How to choose between gradient concentration and fixed concentration? What is the minimum molecular weight of protein separated by precast gel?
A: The size of protein separation by the precast gel is related to its concentration, and the corresponding separation range is shown in the figure above.
Fixed concentration precast gel has better resolution. Gradient concentration precast gel has a wider separation range and a wider range of applications; Customers should choose the appropriate concentration according to their own needs.
Our 4%-20% gradient precast gel (Cat#36250, Cat#36256) has a wide separation range for proteins as low as 3.5kDa.
Figure 3. Gradient precast gel data
Q3: Does the precast gel have to be equipped with your special buffer? Can you make your buffer?
A: YEASEN special buffer (Cat#36236, Cat#36237) and precast gel applicability are better. Do not mix with other buffers. The buffer can be prepared by purchasing reagents, but customers need to use our formula, which can be found in the relevant precast gel instructions.
Q4: How to distinguish between separating gel and concentrated gel? Is it necessary for sectional voltage electrophoresis?
A: The two components are the same, but the concentration is not the same, the naked eye can not distinguish; The height of concentrated glue is 1.5 cm.
No, electrophoresis at 150 V is recommended, and 30-40 minutes is required to complete the experiment.
Q5: Is your precast gel suitable for all kinds of electrophoresis tanks in the market?
A: Yes, our precast gel is suitable for all kinds of electrophoresis tanks in the market,
Bio-rad Mini-Protean (II/3 /Tetra System) and Tanon VE180 (Note the reverse assembly of sealing strip);
Life Technology Novex Mini-cell (used with special baffle);
Other gel plate width in 10 cm electrophoresis tank.
The products provided by Yeasen are as follow:
Table 2. Product details
|Precast Protein Plus Gel, 8%, 10 wells, Hepes-Tris (Inquire)||36245ES10||10 gels/box|
|Precast Protein Plus Gel, 10%, 10 wells, Hepes-Tris (Inquire)||36246ES10||10 gels/box|
|Precast Protein Plus Gel, 12%, 10 wells, Hepes-Tris (Inquire)||36247ES10||10 gels/box|
|Precast Protein Plus Gel, 15%, 10 wells, Hepes-Tris (Inquire)||36248ES10||10 gels/box|
|Precast Protein Plus Gel, 4-12%, 10 wells, Hepes-Tris||36249ES10||10 gels/box|
|Precast Protein Plus Gel, 4-20%, 10 wells, Hepes-Tris||36250ES10||10 gels/box|
|Precast Protein Plus Gel, 8%, 15 wells, Hepes-Tris (Inquire)||36251ES10||10 gels/box|
|Precast Protein Plus Gel, 10%, 15 wells, Hepes-Tris (Inquire)||36252ES10||10 gels/box|
|Precast Protein Plus Gel, 12%, 15 wells, Hepes-Tris (Inquire)||36253ES10||10 gels/box|
|Precast Protein Plus Gel, 15%, 15 wells, Hepes-Tris (Inquire)||36254ES10||10 gels/box|
|Precast Protein Plus Gel, 4-12%, 15 wells, Hepes-Tris||36255ES10||10 gels/box|
|Precast Protein Plus Gel, 4-20%, 15 wells, Hepes-Tris||36256ES10||10 gels/box|
|Precast Running Buffer, 2 L (Powder)||36257ES05||1 packs|
|Precast Running Buffer for Native PAGE, 2 L (Powder)||36258ES05||1 packs|
|GoldBand Plus 3-color Regular Range Protein Marker (8-180 kDa)||20350ES72||250 μL|
|GoldBand Plus 3-color High Range Protein Marker(25-300 kDa)||20347ES72||250 μL|
|GoldBand 3-color Low Range Protein Marker (2.6-40 kDa)||20344ES72||250 μL|