Sephadex G-150 |
Catalog No.GC26203 |
Separation: Peptides and spherical proteins: 5000-40000; Glucan (linear molecule): 1000 to 150000
Products are for research use only. Not for human use. We do not sell to patients.
Sample solution is provided at 25 µL, 10mM.
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Product Documents
Quality Control & SDS
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Protocol
I. Preparation of fillers
1. Allow the fillers to swell in excess deionized water or buffer at room temperature for 48 hours.
2. Balance the swollen fillers, required buffers and other materials to the experimental operating temperature.
3. Filter and degas all buffers.
Note:
(1) When expanding the fillers, it is recommended to use a ratio of water: fillers = 1:20 times or more, that is, add >20ml of water to 1g of filler, and the water should be excessive to fully swell.
(2) You can also use hot water for expansion, which takes 1-3 hours. After mixing boiling water with gel, cool naturally. No continuous heating is required, and water bath is not allowed.
(3) After expansion, if there is a small amount of floating matter on the upper layer, it needs to be removed.
(4) The fillers themselves do not need to be degassed and cannot be ultrasonically treated.
II. Column loading
1. Check all parts of the chromatography column, especially whether the filter screen, sealing ring, and screw plug are tightly assembled, and whether the glass tube is clean and intact.
2. Moisten the inside and bottom of the column with water or buffer and maintain a small liquid level to remove all bubbles.
3. Use a glass rod to guide the homogenate along the inner wall of the column and pour it into the column at once. Do not generate bubbles during the column filling process.
4. Open the column outlet to allow the gel to settle freely in the column and connect the top of the column.
5. Turn on the peristaltic pump (constant flow and pressure must be maintained) and allow the buffer to flow at a flow rate 1.33 times the flow rate during use to stabilize the column bed (note that the pressure should not exceed the maximum pressure resistance of the filler).
Note:
(1) The column filling step is very critical. When pouring the gel, it is required to add the uniform gel to the required column bed height without interruption, otherwise stratification or ripples will occur.
(2) If stratification or ripples are found after the gel is poured, the column needs to be refilled to avoid affecting the chromatography effect.
(3) When making a large gel column, it is often difficult to tell with the naked eye whether the gel is uniform. It is recommended to use some colored macromolecules, such as blue dextran-2000 or cytochrome C, to pass through the gel column before use to observe whether the color bands formed are neat. If the bands are skewed, the column should be reloaded until the requirements are met.
(4) It is recommended to set the flow rate during use to the natural gravity flow rate, that is, the flow rate at which the mobile phase descends with gravity without connecting the peristaltic pump and opening the outlet at the bottom.
(5) The choice of column height is related to the separation requirements. The higher the column, the better the separation effect. However, if the column height is too high, it will cause greater back pressure, which is recommended to be avoided as much as possible.
III. Balance
Before loading the sample, the column needs to be balanced for at least 5-10 column volumes until the filler liquid level does not drop and the recorder baseline becomes stable.
Note: The pH value and conductivity value of the effluent are equal to the pH value and conductivity value of the buffer loaded on the column.
IV. Loading
1. The sample must be completely dissolved. Insoluble matter in the sample will clog the gel, so the sample must be centrifuged or filtered before loading. It is recommended to use a 0.45μm filter membrane for filtration. If the sample is polysaccharide with high viscosity, it needs to be diluted.
2. The loading amount of gel filtration cannot exceed 5% of the column volume.
3. If it is the first loading, the loading amount is recommended to be controlled at 1-2% of the column volume, which can be adjusted according to the separation situation.
4. The desalting loading amount can reach 20% of the column bed volume.
5. Difficult to separate substances must have a certain column height and flow rate control, and the height-to-diameter ratio is 5:1 during desalting.
V. Elution
1. The eluent can use salt-free water or the buffer used when loading the column. The elution buffer is not fixed. The most basic eluent is sterile water, which can be adjusted according to actual needs.
2. The elution buffer should not contain high-viscosity reagents.
3. Flow rate:
(1) When connected to a peristaltic pump: Before the pump is connected, the upper end is not sealed, the lower end outlet is open, and the flow rate is recorded. The flow rate of the peristaltic pump is set according to the natural flow rate.
(2) If the peristaltic pump is not connected: The greater the hydrostatic pressure, the faster the flow rate of the eluent. The hydrostatic pressure can be adjusted freely.
VI. Cleaning in Place (CIP)
Wash 2 column volumes with 0.1M sodium hydroxide, and then regenerate with at least 10 column volumes of equilibrium buffer.
Note:
(1) CIP is performed once after the gel is used about ten times to remove precipitation and residual protein in the column.
(2) After multiple uses, the gel particles will gradually settle and compact, resulting in a slow flow rate. Therefore, it should be poured out after a period of use, cleaned, and then reloaded.
(3) Regeneration cleaning can be performed in the column or poured out and regenerated. If it is performed in the column, the flow rate may slow down. In this case, it is recommended to reload the column.
(4) The sodium hydroxide concentration of the in-situ cleaning solution cannot be higher than 0.1M, and the treatment time cannot be too long, otherwise the packing will be damaged.
VII. Storage
1. Untreated packing should be stored in a sealed container at room temperature.
2. After use, the packing should be thoroughly rinsed with pure water to remove the salt, and finally sealed in 20% ethanol and stored at 4°C.
3. If the experimental interval is short and it needs to be used again the next day, it can be directly stored in the mobile phase.
4. If bacteria grow, it is not recommended to continue using it.
Cas No. | SDF | ||
Formula | M.Wt | ||
Solubility | Storage | RT | |
General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
||
Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. |
In vivo Formulation Calculator (Clear solution)
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Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )
Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
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