Choice of Gel Columns

When choosing a gel column, in terms of appearance, texture and performance, the selected column should be transparent and smooth, with a consistent inner diameter, pressure-resistant and anti-corrosion, and the column material should be biocompatible, and have good physical and chemical resistance and stability. The general column materials are mostly glass or plexiglass. When using steel columns, special attention should be paid to anti-corrosion. In addition, the column terminal must have a collector and a filter screen, and the column bottom plate should be pressure-resistant and not easy to be blocked. The dead volume should be less than 0.001Vt. If the dead volume is too large, the separated components may be remixed, resulting in tailing of elution peaks and reduced resolution. The bottom plate filter media should be new and have meshes that can fuse with the gel. Avoid contact with the mesh and the surface of the filter media, as fingerprints can degrade the separation of the sample. All parts must be thoroughly cleaned when packing or repacking the column.

The length and inner diameter of the gel column mainly depend on the sample volume and resolution requirements. The length of the gel column has a great influence on the resolution. The resolution of the long column is higher than that of the short column, but too long will cause unevenness in the column and the flow rate is too slow. The length of the column generally does not exceed 100cm. At the same time, it is necessary to have an appropriate inner diameter of the column. If the inner diameter is too thick, serious lateral diffusion of protein samples will occur. If the inner diameter is too thin, the flow velocity near the inner wall of the column will be greater than the flow velocity in the center, resulting in a “wall effect”, which affects the separation speed and effect. . The length of the gel column usually used is 25-70cm, the inner diameter is 4-16mm, and the H/D (height to diameter ratio) is generally between (25:1)-(100:1). The H/D of the gel column used for group separation can be relatively low. The H/D can be between (30:1)-(100:1) during fractionation. The desalting column has a higher resolution due to the higher requirements. Low, generally use short columns, H/D is mostly between (5:1)-(25:1). If the molecular weights of the proteins to be separated are quite different, a column with H/D=(15:1)-(50:1) can be used, and the column volume should be larger than 4-15 times the sample volume. If the molecular weights of the proteins to be separated are different If it is smaller, use a thin column with H/D=(20:1)-(100:1), and the column volume should be larger than 25-100 times the sample volume. When purifying proteins, the H/D of the column should be (20:1)-(40:1) for good resolution.

In addition, the size of the gel particles should also be considered when choosing a column. For packing small particle gels, use a larger diameter column. When packing with coarse particles, use a smaller diameter column.

Pretreatment of Gels

Commercially available gels are generally available as dry powder granules (eg Tandex) or water suspensions (eg Tanrose CL). Some gels that should not be stored in a dehydrated and dry state (such as agarose gels) should be stored in an aqueous solution containing preservatives. These gels do not need to be swollen before use. In addition, porous silica gel and porous glass do not require swelling treatment. In order to minimize the influence of different solvents on the column bed volume and obtain the ideal separation effect, the dry gel should be slowly poured into the eluent with a volume of 5-10 times the dry gel volume to fully swell. If the swelling is insufficient, effective separation will not be achieved and the gel column will rupture during use.

After selecting the type of gel used according to the molecular weight and resolution requirements of the protein sample, the amount of dry gel used is estimated based on the column volume and the water absorption properties of the gel. Because the gel has a certain amount of loss during pretreatment and experimental operation, it should be 10%-20% higher than the theoretical calculation value when actually weighed.

Different types of gels require different swelling times. Generally, a gel with a smaller size and a lower exclusion limit has lower water absorption and a shorter swelling time. It takes 3-4 hours at 20 °C. A gel with a larger size and a higher exclusion limit Glue has a large amount of water absorption, so the required swelling time is also longer, and it takes more than ten hours to dozens of hours at about 20 °C.

Heating swelling is a commonly used gel pretreatment method, that is, the weighed dry gel powder is dissolved in the eluent and gradually heated to near boiling. This method can greatly shorten the swelling time, which can generally be completed within 1-2 hours, and can play a triple effect of swelling, degassing and sterilization. During the gel swelling process, stirring should be continued slowly, but not vigorously, because it is easy to cause the breakage of the gel particles, and eventually the fine particles will block the gel column and affect the flow rate and separation effect. In addition, heating and swelling in acid or alkali should be avoided as much as possible to prevent damage to the mesh structure of the gel.

After fully swollen, the gel homogenate should be suspended to see if there are impurities or uneven fine particles in the supernatant. It is very important to remove air bubbles from the swollen gel, otherwise it will affect the separation effect. Generally, air bubbles can be removed by vacuum pumping or heating and boiling. Due to the high price of gels, losses and waste should be minimized during use, and the remaining gels should be stored for later use.

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