This Article will briefly explain the application of Gel Filtration. We hope it will help you to know more about gel filtration. So let’s start…
1. Purification of biological macromolecules
Gel filtration is based on different molecular weights for separation. Because of its separation characteristics, and its advantages such as simplicity, convenience, and no change to the biological activity of the sample, gel filtration has become an important way to purify biological macromolecules. Means, especially for molecules of different sizes but similar physical and chemical properties, it is difficult to separate them by other methods, and gel filtration is undoubtedly a suitable method, such as the separation of polymers with different degrees of polymerization.
2. Molecular weight determination
The operation of gel filtration to determine the molecular weight is relatively simple, and the amount of sample required is small. It is an effective method for preliminary determination of protein molecular weight. The disadvantage of this method is that the accuracy of the measurement results is affected by many factors. Since this method assumes that neither the standard nor the sample has an adsorption effect on the gel, if the standard or sample has a certain adsorption effect on the gel, the measurement error will be relatively large. The condition for the establishment of the calculation formula is that the protein is basically spherical, which does not hold for some elongated proteins such as fibrin. In addition, due to the strong hydration effect of sugar, when gel filtration is used to measure glycoprotein, the measured molecular weight is too large, and when ferritin is measured, the measured value is found to be too small.
3. Desalination and removal of small molecular impurities
Desalination and removal of small molecular impurities by gel filtration is a simple, effective and rapid method. It is much faster than the general method of dialysis for desalination, and generally does not cause large dilution of the sample, and biomolecules are not easily denatured. . Generally, Welch Tandex G-25 is commonly used, and there are many kinds of desalination column products sold, which are easy to use and can be used multiple times.
4. Depyrogenation material
Heat source substances refer to certain polysaccharide protein complexes produced by microorganisms that make the human body heat up. They are a kind of material with a large molecular weight. Therefore, the exclusion effect of gel filtration can be used to compare these macromolecular heat source substances with other relative molecular weights. Small substances separate. For example, gel filtration is a simple and effective method to remove water, amino acids, and some pyrogens in injections.
5. Concentration of the solution
The macromolecule sample solution can be concentrated by using the water absorption of gel particles. For example, the dry Tandex (coarse particles) can be added to the solution. Tandex can absorb a lot of water. Small molecules in the solution will penetrate into the gel hole, while the macromolecules will be excluded. A concentrated sample solution can be obtained by removing gel particles by centrifugation or filtration. This concentration method basically does not change the ionic strength and pH value of the solution.
6. Protein refolding
In order to reduce the aggregation reaction at high concentrations, gel filtration chromatography technology can also be used to refold the protein in vitro. The isolation effect of the chromatographic medium reduces the aggregation caused by the interaction between the proteins, so that the refolding concentration and the refolding rate are greatly improved. At the same time, the protein can also be purified to a certain extent through gel filtration chromatography itself.
Two important factors affect the yield of renaturated protein by gel filtration chromatography: the first is the loading of the protein under denaturing conditions, and the second is the change of protein structure in the renaturation buffer. In addition, the amount of protein loaded will also affect the yield of protein renaturation, because the top of the chromatography column will cause structural aggregation due to the increase in the amount of sample loaded.
In order to improve the efficiency of protein renaturation, a gradient gel filtration chromatography renaturation method was developed. Pre-set the denaturant concentration gradient in the chromatographic column. When the renatured protein enters the column, the apparent molecular weight of the protein is much larger than the denaturant, so the protein passes through an environment where the denaturant concentration gradually decreases. Ground folding and renaturation effectively reduces the formation of aggregates and separates aggregates from folded proteins.
Linear gradients may often be encountered in gel filtration chromatography, but sometimes some proteins may be unstable at certain denaturant concentrations, and these concentration processes need to be crossed as quickly as possible during the gradient process; sometimes the rate of protein folding is limited in certain At the denaturant concentration, this period of time needs to be increased at this time, so different types of gradient forms can be designed in gel filtration chromatography to meet the needs of different protein renaturation.
Welch Materials Gel Filtration Packing:
- Tandex G series and LH20 multi-mode gel filtration series
- SuperTandex Prep Grade series
- Tanrose Fast Flow series
- Tanrose/Tanrose CL series
If you have any problem or require further information, please contact firstname.lastname@example.org.