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    Home > Biochemistry News > Biotechnology News > The concept of gel analysis and its principles.

    The concept of gel analysis and its principles.

    • Last Update: 2020-10-20
    • Source: Internet
    • Author: User
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    types and effects of gelics) and gelication definitions
    gel analysis, also known as molecular sieve
    filtration
    and stratitography, etc. Its outstanding advantage is that the gel used in layering belongs to the inert carrier, without charge, weak adsorption force, relatively mild operating conditions, can be carried out in a fairly wide temperature range, do not need
    organic
    solvent, and the separation of the chemical properties of the components to maintain unique. It has a good separation effect on polymer substances. Gel analysis is a technique for separation according to
    protein
    molecular weight, also known as gel filtration, molecular sieve or de-dissosis.principle of gela single gel bead itself is like a "sieve". The size of the perforation of different types of gels is different. If the gel is loaded into a column long enough to form a gel column. When protein samples containing different sizes are added to the gel column, proteins with a smaller average aperture than the gel beads will continue to penetrate the inside of the beads continuously, and such small molecules not only have a long movement distance, but also by the resistance from the inside of the gel beads is also very large, so the smaller the protein, the longer it takes to wash them off the column! Only a small number of apertures in the gel are acceptable for large proteins. As a result, large proteins are first washed away through the gap between the gel beads. The choice of gel aperture size for gel filtration depends mainly on the molecular weight of the protein to be purified.gel layering classification glucosaccharide gel refers to a gel made from natural polymer-glucosaccharides in association with other crosslinking agents. Glucosin 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 2 ethylene propane. The main model of Sephadex is the G-10 to G-200, followed by 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 5mL/g of 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 8×10..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 to 10. Strong acid solutions and oxidants cause crosslinked glycoside bonds to break, so avoid Sephadex's contact with strong acids and oxidants. Sephadex is stable at high temperatures and can be boiled for disinfection, with no significant effect on the structure and performance of the gel at 40 min at 100 degrees C. Sephadex is weakly acidic because it contains hydroxyl groups, which makes it possible to adsorption from some charged groups (especially alkaline proteins) in the isolates. However, under the condition that the ion strength is 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 solution, 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 relatively poor mechanical stability, its pressure resistance and high resolution fine particles require slower flow, so fast and efficient separation is not possible. In 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, separation of fat-soluble substances, such as cholesterol, fatty acids
    hormone
    and so on..Sephacryl is a cross-link between glucosaccharides and methicilline (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, far beyond the scope of Sephadex. 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 its higher chemical and mechanical stability: Sephacryl rarely dissolves or degrades in a variety of solvents, can be used as a sequestration solution with a variety of detergents, terse, radon, etc., high temperature resistance, Sephacryl stable work pH is generally 3 to 11. In addition, Sephacryl's mechanical properties are better, can be washed away at a higher flow rate, more pressure-resistant, higher resolution, so Sephacryl compared to Sephadex can achieve relatively fast and higher resolution separation.the gel,geldifferent models of gel according to the experimental purpose. If the purpose of the experiment is to separate the large and small molecular substances in the sample, due to their significant differences in distribution coefficients, this separation is also known as group separation, generally optional Sephadex G-25 and G-50, for small peptides and low molecular weight substances (1000-5000) desalination can be used Sephadex G-10, G-15 and Bio-Gel-p-2 or 2. If the purpose of the experiment is to separate some substances in the sample with a relatively similar molecular weight, this separation is also known as graded separation. Generally selected resistance limit is slightly greater than the sample of the highest molecular weight substances gel, the layering process of these substances can be in varying degrees deep into the gel inside, due to Kd different, and finally get separated.According to experience, when the groups are separated, most of them use 2-30cm long layering column, grade separation, generally need 100cm long column, its diameter in the 1-5cm range, less than 1cm to produce pipe wall effect, greater than 5cm is a serious dilution phenomenon. The ratio of length L to diameter D L/D is generally between 7-10, but the slow-moving substance should be between 30-40.the preparation of the gel columngel model is selected, the dry gel particles are suspended in 5-10 times the amount of distilled water or sequestration liquid fully soothing, after the swelling will be very fine particles poured out. Natural swelling takes a long time,
    heating
    can accelerate the swelling, that is, in the boiling water bath will gradually heat up the wet gel slurry to near boiling, 1-2 hours can reach the full expansion of the gel. Heating saves time and can be disinfected.gel filling: the layer column and the ground perpendicularly fixed to the shelf, the lower flow outlet with clip clamping, the top of the column can be installed with a stirring device of a larger container, the column is full of seventing liquid, the gel into a thinner The thin slurry head is stored in a container at the top of the column, and then, with a slight stirring, the gel sinks into the column so that the gel grain level rises until the desired height, remove the column top device and gently cover the surface of the gel bed with the appropriate filter sheets. Place for a while before you begin to flow equilibrium, and the flow rate should be lower than the flow rate required for layering. Gradually increase the flow rate of layering in the process of equilibrium, and must not exceed the final flow rate. Balance the gel bed overnight, before use to check whether the layering bed is uniform, whether there are "patterns" or bubbles, or add some colored material to observe the movement of the ribbon, such as narrow, uniform flat description of the performance of the column is good, the ribbon appears distorted, scattered, widening must be re-posted.the gel bed is balanced withsample and wash-out, leave several liters of semen at the top of the bed to saturate the gel bed and add the sample with a dropper. The general sample volume is not greater than 5%-10% of the total gel bed volume. The sample concentration is not related to the distribution coefficient, so the sample concentration can be increased, but the viscosity of the solution will increase with the concentration, so that the relative viscosity of the sample and the wasculation solution should not exceed 1.5-2. After the sample is added to open the flow outlet, so that the sample seeps into the gel bed, when the sample liquid surface is exactly the same as the surface of the gel bed, and then add a few milliliters of secrubbing liquid in the washing tube wall, so that it all into the gel bed, the layering bed and the desalination tank and collector connected, pre-designed flow rate, and then part of the collection of sempulation liquid, and each fraction to do qualitative, quantitative determination.reuse, gel recovery and preservation of gel columnscan be used repeatedly after a column installation, without special treatment, and does not affect the separation effect. In order to prevent gel contamination, 0.02% of sodium laminated nitrogen can be added after one layering, and the antibacterial agent should be removed before the next layering, so as not to interfere with the determination of the ebony.If it can no longer be used can be recycled, the general method is to rinse the gel with water clean and dry, in turn with 70%, 90%, 95% ethanol dehydration balance to ethanol concentration of more than 90%, filter dry, and then use ether to wash away ethanol, filter dry,
    drinding
    preservation. Wet preservation method is to add antibacterial agent or water to the gel pulp flushed to neutral, sealed after autocultic preservation.application of gel lithography (1) desalination: low molecular weight impurities in polymer (e.g. proteins, nucleic acids, polysaccharides, etc.) solutions can be removed by gel lithography, an operation known as desalination. This method desalination operation is simple, fast, proteins and enzymes are not easily denatured in the desalination process. The applicable gels are SephadexG-10, 15, 25 or Bio-Gel-p-2, 4, 6. The ratio of column length to diameter is 5-15, the sample volume can reach 25%-30% of the volume of the column bed, in order to prevent protein desalination after the solubility is reduced will form precipitation adsorption on the column, generally with ammonium acetate and other volatile salt buffer to balance the column, and then add samples, and then wash away with the same buffer, collected washesis with frozen drying method to remove volatile salts.(2) for separation purification: gel lithography has been widely used in enzymes, proteins,
    amino acids
    , polysaccharides, hormones, alkaloids and other substances separation purification. The gel has a strong adsorption force to the heat and can be used to remove the heat-ion-free water from thermogen preparation injection water.(3) Determining the molecular weight of polymeric substances: using a series of known molecular weights of
    systricts
    into the same gel column, under the same conditions layering, recording the volume of each minute of the composition of the escape, and the volume of the volume of the logaturs of the molecular weight, in a certain molecular weight range can be a straight line, that is, the standard curve of molecular weight. When determining the molecular weight of unknown substances, the sample can be added to the gel column of the standard curve, according to the volume of the material's escape, the molecular weight of the standard curve can be found.(4) Concentration of polymeric solutions: SephadexG-25 or 50 drys are usually put into a thin polymer solution, where water and low molecular weight substances enter the pores inside the gel particles, while polymeric substances are discharged outside the gel particles, and then centrifuged or filtered to separate the swollen gels, resulting in a concentrated polymer solution
    .
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