The regenerated cellulose in Cellu·Sep® is derived from cotton: Cotton linters are dissolved in a solution and spread into flat sheets or extruded into tubes. The material is treated with glycerin to prevent the pores from collapsing and air dried at a certain temperature and pressure to form a rigid membrane. Cellu·Sep regenerated cellulose membrane has a symmetric pore structure which allows small molecules to migrate in either direction, making it ideal for experimental purposes.
Low-molecular weight salts and buffers (e.g.,Tris·Cl and KPO4) equilibrate within 3 hours with stirring. Equilibration times for viscous samples will be longer.
Change the dialysis buffer as necessary. Usually two dialysis buffer changes are sufficient. When CsCl is removed from equilibrium density gradient-banded DNA, two equilibrations against a 1000-fold volume excess of buffer will decrease CsCl concentration 106-fold, to a still-significant 5 µM, and it may be necessary to change buffer a third time.
Cellu·Sep membranes exhibit a very low non-specific protein adsorption when compared with competitive membranes. Cytochrome C solutions exhibit a large adsorptive loss with competitive membranes following a five fold concentration of 150µg/ml solution.
In addition to very low non-specific adsorption the Cellu·Sep membranes have excellent chemical resistance against organic solvents. Click here to skip to chemical compatibility charts located in this document.
If glycerol, sulfur compounds, or small amounts of heavy metals will interfere with subsequent steps, the membrane should be prepared as described below. For convenience, pre-treated, highly clean Cellu·Sep H1 High Grade wet membranes, designed for sensitive applications, may also be used.
1. Wearing gloves, cut the dialysis membrane into the desired length and soak in distilled water for 15 minutes.
2. Heat the pre-cut membrane for 30 minutes, while stirring, to 80 °C in a large volume of 10 mM sodium bicarbonate.
3. Transfer the membranes into a 10 mM Na2EDTA solution and soak for 30 minutes.
4. Replace solution with distilled water and stir for 30 minutes at 80 °C.
5. Allow membrane to cool down, and store in a refrigerator in a 0.05% sodium azide solution, or a 0.10% sodium benzoate solution. Alternatively, a 20-50% ethanol solution may be used. Tubing must always remain submersed.
6. Before use, wash tubing inside and out with distilled water and condition in dialysis buffer (if necessary, tubing may be sterilized).
7. Secure clamp on one end of membrane. Buffer or water should be placed inside the bag to ensure integrity of the seal. Check integrity of the tubing and clamps.
8. Pour out test solution and load sample (For concentrated salt samples, leave space in the tubing to allow for net flow of water into the sample and to prevent tubing from bursting.)
10. Change the dialysis buffer as necessary. Usually two dialysis buffer changes are sufficient. When CsCl is removed from equilibrium density gradient-banded DNA, two equilibrations against a 1000-fold volume excess of buffer will decrease CsCl concentration 106-fold, to a still-significant 5 µM, and it may be necessary to change buffer a third time.
11. Remove dialysis tubing from buffer, remove clamp from one end, and remove sample with a pipet.
The common method of membrane sterilization is exposure to ethylene oxide gas. Alternative sterilization methods are gamma irradiation and steam autoclaving. Several published abstracts illustrate a decrease in permeability after a sterilization process, depending upon the applied method. Below are some of the techniques described.
Suggested preparation of membranes before sterilization is to soak the membrane for 30 minutes in distilled water. After washing, the membrane might be stored in a 0.85% sodium chloride solution containing 0.1% formaldehyde.
Place the prepared membrane in an open polyethylene bag in a vacuum oven. Evacuate and fill the oven with a gas mixture of 20% EtO/ 80% CO2 by a total pressure of 1 atm. Treat the membrane for 5 hours at 40 °C. Evacuate the sterilizing gas and admit 50% of relative humidity of air. A slight reduction, approximately 10% permeability characteristic, has been reported with the use of this EtO method.
Seal the membrane in a polyethylene bag and expose to a gamma ray source for a total dose of 2.5 Megarads. During exposure the temperature should not increase to more than 10 °C. The permeability characteristic after the treatment is approximately 75%.
The length of the autoclaving cycle should be kept as short as possible. Membranes may be safely autoclaved at 121 °C at 100 kPa (1 bar) for 10 minutes if submersed in distilled water. They should not be permitted to dry out afterwards. Dry heating over a period of 48 hours at 80 °C drops the permeability characteristic to about 50%.
Membranes, when wet, are most susceptible to microbes and fungi. Such microbial growths will impair the dialysis properties of the material and cause decreased yield and infection of the sample. Therefore, tubing should not be left without satisfactory protection against bacteria and fungi.
Sodium azide and sodium benzoate are commonly used preservatives for serological material. A concentration of 0.05-0.10% sodium azide is recommended for this purpose. However, a considerably lower concentration, around 0.02%, seems to give satisfactory protection, although streptococci growth may occur at this concentration.
Sodium Azide (NaN3) is a standard preservative for laboratory reagents. Although NaN3 is considered highly toxic its LD50 in rats is 45 mg/kg orally.
MFPI recommends the following Sodium Benzoate (C7H5NaO2) solution in addition to Sodium Azide (NaN3) for suppressing microbiological growth in wet stored dialysis membranes. Sodium Benzoate is considerably less toxic than Sodium Azide (LD50 in rats of C7H5NaO2 is 4.07 g/kg orally).
Sodium Benzoate is commonly used as a preservative in pharmaceuticals and in food products (not more than 1 in 1000 parts being permitted). Although its preservative effect is best exhibited in slightly acidic media; in neutral media it provides satisfactory protection of the membrane.
Reagent: C7H5NaO2, Molecular Weight: 144.04
Dissolve 7.2 grams of C7H5NaO2 in 100 ml of water. The solution may be brought to a boil to speed the dissipation of the C7H5NaO2.
All Cellu·Sep dialysis membranes are packaged in unique dispenser boxes to facilitate ease in handling and measuring. The membranes are enclosed in polyethylene bags, containing desiccant pouches to prevent excess moisture and reduce the risk of contamination. The membranes contain glycerol, added as a humidifier and plasticizer to maintain product integrity.
To prevent drying and subsequent brittleness, it is vital for the tubing to be stored properly. Moisture loss can cause loss of membrane flexibility and result in pinholes during handling. In order to avoid this possibility, it is recommended that unused tubing be stored in the original polyethylene bag or any other air tight moisture-proof container in a cool place (refrigerator), in a minimum of 35% relative humidity.
Regenerated cellulose has excellent resistance against organic solvents and is predominantly used for the filtration of non-aqueous liquids with which other materials are not compatible. Chemical compatibilities can be influenced by various factors. Therefore, we recommend that you confirm compatibility with the liquid you wish to use by performing a trial run before you begin with actual dialysis.
G - Compatible
F - Limited compatibility (swelling or shrinkage may occur)
N - Not compatible
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