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    Home > Biochemistry News > Biotechnology News > Types, operation and application of gel analysis (molecular blocking layering).

    Types, operation and application of gel analysis (molecular blocking layering).

    • Last Update: 2020-10-22
    • Source: Internet
    • Author: User
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    related topics .Gel layering is a kind of layering technique that uses the difference of the molecular weight of the separated
    protein
    , also known as gel
    filtration
    or molecular sieve or molecular blocking. The greatest advantage of molecular desetosis is that the conditions required for the experiment are extremely mild.Introduction to gelgel chromatography, also known as gel chromatography, molecular sieve chromatography, gel filtration, gel permeation, chromatography, etc. It is a liquid phase
    chromatography method for separating the components in the sample in molecular
    with porous gel fillers as
    phases.
    1959, Porath and Flodin first used a porous polymer-crossed glucosaccharide gel as a column filler to separate samples of different molecular weights in the aqueous solution, called gel filtration.
    In 1964, Moore developed a cross-linked polystyrene gel with different apertures that allows separation in organic solvents, known as gel osmosis (gel layering with flow phases for organic solvents is commonly referred to as gel osmosis). Subsequently, this technology has been continuously improved and developed, is widely used in
    biochemistry,
    , polymer chemistry and many other fields.gel layering is a commonly used separation method in biochemistry, it has the advantages of simple equipment, easy operation, high sample recovery rate, good experimental repeatability, especially not changing the biological activity of samples, so it is widely used in protein (including enzymes), nucleic acids, polysaccharides and other biological molecules separation purification, but also used in protein molecular weight determination, desalination, sample concentration and so on.principle of gelgel is to separate and purification according to the physical properties of molecular size. The fixed phase of gel layering is an inert beaded gel particle, which has a three-dimensional mesh structure inside, forming many holes. When samples containing components of different molecular sizes enter the gel column, the components spread into the holes of the fixed phase, and the degree of diffusion of the components depends on the size of the holes and the size of the component molecules.
    molecules with a large aperture can not spread to the inside of the hole, completely blocked outside the hole, can only flow downwards with the flow phase in the space outside the gel particles, they experience a short process, flow speed, so first flow out;
    the degree of penetration depends on the size of their molecules, so the time they flow out is somewhere in between, the larger the molecule, the smaller the part, the later it flows out. Thus, after the sample has been analyzed by gel, the components flow out in order of molecules from large to small, thus achieving the purpose of separation.1, the volume of external water, the volume of inner water, the volume of the substate, the volume of the column bed, the volume of the exoded water the volume of the outer water refers to the volume of the space around the gel particles in the gel column, that is, the volume of the liquid flow phase between the gel particles. The volume of inner water refers to the volume of holes in gel particles, and the fixed volume in gel analysis refers to the volume of inner water. The mass of the substate refers to the actual skeleton volume of the gel particles. The volume of the column bed refers to the total product that the gel column can hold.
    volume is the volume of the amount of escape fluid required to unwash a certain part of the sample. We set the column bed volume is Vt, the outer water volume is Vo, the internal water volume is Vi, the substation volume is Vg, then there are: Vt- Vo-Vi-Vgbecause Vg is relatively small and negligible, then there is: Vt-Vo-Vi set-up wash-out volume is Ve, Ve is generally between Vo and Vt. For large molecules that are completely blocked, because they do not enter the gel particles, but only exist in the flow phase, so their excreted volume Ve-Vo, for fully permeable small molecules, because it can exist in the entire volume of the gel column (ignoring the gel itself volume Vg), so its excreted volume Ve-Vt.
    between the two molecules, and their escape volume is somewhere in between. Sometimes there may be a Ve? Vt, which is caused by the adsorption of this molecule and the gel.Vt can be measured by adding a certain amount of water to the predetermined marker of the column and then measuring the volume of the water. The volume of outer water Vo can be determined by measuring the excret volume of a fully blocked large molecular substance, and blue glucosaccharose-2000 is commonly used as a substance to determine the volume of outer water. Because of its large molecular weight (2 million), it is blocked in various types of gels, and it is blue and easy to observe and detect.2, distribution factor distribution coefficient refers to the concentration ratio of a certain part in the fixed phase and the flow phase. For gel analysis, the distribution coefficient essentially represents the distribution relationship between the volume of water in the inner part and the concentration in the volume of external water. 3, the exhaust limit the exhaust limit refers to the molecular weight of the smallest molecule that cannot enter the hole of the gel particle. All molecules greater than the resistance limit cannot enter the gel particles and flow directly out of the gel particles, so they are also first excreted.
    the maximum molecular weight that a gel can effectively separate, and molecules greater than the resistance limit of this gel cannot be separated with this gel. For example, the Sephadex G-50 has an resistance limit of 30,000, which means that molecules with a molecular weight greater than 30,000 are washed out directly from the gel particles. 4, graded separation range graded separation range represents a gel applicable separation range, for the molecular weight in this range of molecules, with this gel can obtain a better linear separation. For example, Sephadex G-75 has a graded separation range of 3,000-70,000 globulins, which indicates that globulins with a molecular weight in this range can be better separated by Sephadex G-75. It should be noted that for the same type of gel, the graded separation range of globulin and line protein is different. 5, absorption rate and bed volume water absorption rate refers to the volume or weight of 1 g of dry gel absorbing water, but it does not include water adsorbed between particles. So it does not represent the volume behind the gel column. The bed volume refers to the final volume after 1g of dried gel absorbs water. 6, gel particle size the gel used for layering is generally spherical, the size of the particles is usually expressed as mesh or particle diameter (m). The resolution and flow rate of the column are related to the size of the gel particles. The grain is large, the flow rate is fast, but the separation effect is poor, the particle is small, the separation effect is good, but the flow speed is slow. Generally more commonly used is 100-200 eyes. types and properties of gels gels are available, with commonly used gels being a crosslink between glucosamine gels (dextran), polyacrylamide gels,
    agar
    sugar gels, and polyacrylamides and agalipose. There are also porous glass beads, porous silicones, polystyrene gels and so on. This is described separately below. 1, glucosaccharide gel glucosaccharide gel refers to the natural polymer-glucosaccharide and other cross-linked agents of the gel. Glucosaccharin gels are mainly produced by Pharmacia Biotech. There are two common categories, Sephadex and Sephacryl. most common of the glucosaccharide gels is the Sephadex series, which is a cross-link between glucosaccharides and 3-chlorine-1,2 epoxy propane (crosslinking agents), which is controlled by a percentage of epoxy chloropropane. The main model of Sephadex is the G-10 ? G-200, followed by the number is the gel's water absorption rate (in ml / g dry glue) multiplied by 10. A Sephadex G-50, for example, indicates a water absorption rate of 5 ml/g dry glue.
    Sephadex is very hydro-hydro, very easy to expand in water, different models of Sephadex absorbent rate, their hole size and separation range is also different. The larger the number, the greater the resistance limit and the greater the separation range. The G-200 with the largest resistance limit in Sephadex is 6?105.
    Sephadex is stable in aqueous solutions, salt solutions, alkali solutions, weak acid solutions, and organic solutions and can be reused multiple times. Sephadex stable work pH is generally 2-10. Strong acid solutions and oxidants dehydrate crosslinked glycoside bonds, so avoid Sephadex from contact with strong acids and oxidants.
    Sephadex stabilizes at high temperatures and can be boiled and disinfected at 100 ? The lower 40 min C has no significant effect on the structure or performance of the gel. Sephadex is weakly acidic due to its hydroxyl clumps, which makes it possible to adsorption from some charged groups in the isolate, especially alkaline proteins.
    but generally under conditions with ion strength greater than 0.05, there is little adsorption. Therefore, in the gel analysis experiment with Sephadex often use a certain concentration of salt solution as an enchantment, so as to avoid Sephadex and protein adsorption, but should be aware that if the salt concentration is too high, will cause a large change in the size of the gel column bed.
    Sephadex has a variety of particle sizes (generally coarse, medium, thin, ultra-fine) to choose from, generally coarse particles flow fast, but the resolution is poor; The particle size is to be selected according to the separation requirements. Sephadex's mechanical stability is relatively poor, it is not pressure-resistant, and fine particles with high resolution require slower flow, so fast and efficient separation is not possible. addition, Sephadex G-25 and G-50 were added to the hydroxypropyl group reaction, forming LH-type alkylized glucosaccharide gel, the main models are Sephadex LH-20 and LH-60, suitable for organic solvents as the flow phase, the separation of fat-soluble substances, such as cholesterol, fatty acids
    hormone
    and so on. . Sephacryl is a combination of glucosacrylamide (N, N'-methylenebisacrylamide). It is a relatively new type of glucosaccharin gel. The advantage of Sephacryl is that it has a large separation range, and the resistance limit can even reach 108, which is much larger than Sephadex's range. So it can be used not only to isolate general proteins, but also to isolate protein polysaccharies, protons, and even larger viral particles.
    Another advantage of Sephacryl over Sephadex is that it has higher chemical and mechanical stability: Sephacryl rarely dissolves or degrades in a variety of solvents, can be used as a scrub of various detergents, niacin, radon, etc., high temperature resistance, Sephacryl stable work pH is generally 3 to 11. addition, Sephacryl has better mechanical properties, can be washed away at a higher flow rate, is more pressure-resistant, and has a higher resolution, so Sephacryl can achieve relatively fast and high resolution separation compared to Sephadex. 2, polyacrylamide gel polyacrylamide gel is acrylamide (acrylamide) and fork diacrylamide cross-linked. By changing the concentration of acrylamide, the product of different crosslinks can be obtained. Polyacrylamide gels are mainly produced by Bio-Rad Laboratories and are available under the name Bio-Gel P, with the main models bio-Gel P-2? There are 10 species, such as the Bio-Gel P-300, and the numbers behind them basically represent 10-3 of their resistance limits, so the larger the number, the greater the sethicable molecular weight. The main parameters of the various models are shown in the schedule.
    separation range and water absorption rate of polyacrylamide gels are similar to Sephadex. The Bio-Gel P-300 with the largest resistance limit is 4?105. Polyacrylamide gels are stable in aqueous solutions, general organic solutions and salt solutions. Polyacrylamide gels are more stable in acids and between 1 and 10 pH.
    , polyacrylamide gels break down easily under strong alkaline conditions or at higher temperatures. Polyacrylamide gel is very hydro-hydro, basically no charge, so the adsorption effect is small. In addition, polyacrylamide gels do not grow like glucosaccharide gels and agarose gels,
    microbial
    . Polyacrylamide gels may have a slight adsorption effect on aromatic, acidic, and alkaline compounds, which can be avoided with slightly higher ion strength scavenging fluids. 3, agarose gel agar is a natural linear polysaccharose isolated from agar, which is derived from agar removing the charged agar gum. Agarose is a polysaccharose chain alternating between D-half lactose (D-galactose) and 3,6-dehydrated semi-lactose (anhydrogalactose). It's at 100 ? C is liquid,
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