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Sephadex G 25

CAS No. : 9041-35-4

MCE 站：Sephadex G 25

产品活性：Sephadex G 25 是一种葡聚糖层析介质。Sephadex G-25 分离范围为 1-5 kD (球形蛋白)，可用于多肽的分离以及大分子蛋白质的脱盐和缓冲液替换。

研究领域：Others

作用靶点：Others

In Vitro: Guidelines (Following is our recommended protocol. This protocol only provides a guideline, ａnd should be modified according to your specific needs).
1. Swelling: The dry rubber particles need to be swelled for the first time. Place in excess deionized water ａnd swell at room temperature for 3 h, or in a 90°C water bath for 1 h. (forbid to use magnetic stirring during the swelling process to avoid breaking the microspheres)
2. Column packing:
(1) Prepare initial buffer (equilibrium solution) ａnd elution buffer according to the properties of the samples to be separated. In the case of desalting proteins such as proteins, it is generally recommended to use only one buffer with a low salt concentration for sample loading, equilibration ａnd elution in gel chromatography. For example, 50 mM Tris-hydrochloric acid or PBS buffer near neutral; if it is the separation of peptides, 0.15 M NaCl can be appropriately added to shield the interaction between the target ａnd the gel; if it is buffer exchange, ｓelect the target buffer as needed.
(2) Drain the gel, prepare a homogenate with initial buffer (at the ratio of gel:buffer = 3:1) ａnd degas.
(3) Fix the column vertically, wet the bottom end with water or buffer ａnd keep the liquid level for a period.
(4) Guide the homogenate with a glass rod ａnd pour it into the column at one time along the inner wall of the column to allow the gel to settle freely in the column, seal the column with water, ａnd settle overnight.
(5) Connect the movable column head at the top of the column, turn on the peristaltic pump, use the operating flow rate to let the buffer flow through 5 times the column volume, ａnd then use 1.5 times the operating flow rate to flow through 5 times the column volume, ａnd adjust the adapter head to make it as close as possible. Finally, equilibrate the column with 2-3 column volumes of buffer.
Note: Air bubbles cannot be introduced during all operations to ensure the uniformity of the gel.
3. Equilibration: Equilibrate the column with the equilibration buffer at the operating flow rate, ａnd observe the change of the detector until the parameters such as conductivity ａnd pH remain unchanged.
4. Loading: switch the switching valve to load the sample, ａnd the sample load is selected according to the nature of the sample ａnd the amount of the chromatography medium. Generally speaking, the sample load does not exceed 25% of the column bed volume. The samples were prepared with equilibration solution ａnd pretreated by filtration with 0.45 μm microporous membrane.
5. Elution: Switch to elution buffer for constant elution, after peaking, to baseline equilibrium.
6. Regeneration: wash with 0.2 mol/L NaOH or non-ionic detergent for 2 times the column volume; then rinse with deionized water for 3-5 times the column volume.
7. Storage: After use, replace the mobile phase with 20% ethanol ａnd store in a refrigerator at 4°C.
8. In order to obtain a better desalination effect, it is recommended to keep the column heights of different particle sizes at: 30 cm ａnd above for coarse particles, 20 cm ａnd above for medium particles, 10 cm ａnd above for fine particles, 5 cm ａnd above for ultra-fine particles, ａnd The smaller the particle size, the higher the resolution ａnd the slower the flow rate.
9. The amount of sample added should be determined according to the experiment. Generally, the maximum sample load does not exceed 30% of the column bed volume, ａnd 10-20% is the best sample load.

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