SEPHADEX G-100 100 GArticle No: 17006001General Product Information
===============================================ORDERINGPack size Code No.100 g 17-0060-01500 g 17-0060-025 kg 17-0060-0340 kg 17-0060-07
¤ Thousands of documented applications.¤ Proven reproducibility.¤ Excellent recoveries.¤ Economical.
---------------------------------------------------------------------------------Application area (Details) ===============================================* Fractionation and purification of proteins andpeptides.
* Determination of molecular weights.* Determination of equilibrium constants.
---------------------------------------------------------------------------------Autoclaving (Details) ===============================================In wet form (pH 7), at 120°C for 30 min.
---------------------------------------------------------------------------------Calibration (Details) ===============================================Proteins: Use LMW Gel Filtration Calibration Kit.---------------------------------------------------------------------------------Checking the column packing (Details)
===============================================1. Run Blue Dextran at 2 mg/ml.
2. Watch the progress of a zone of this substancethrough the bed:
You should see a discrete horizontal bandtravelling through the column.If not:
A. The problem could be caused by dirty filters.Generated at:
12/09/2002 05:45:57 PMB. More likely:
An uneven bed surface or uneven packing isresponsible, in which case the column must berepacked.
---------------------------------------------------------------------------------Choice of eluent (Details)
===============================================* Eluent composition does not directly influence theresolution which can be obtained:
A. Completely uncharged substances may be elutedwith distilled water.
B. For substances carrying charged groups an eluentcontaining a buffer e.g. Tris-HCl, sodiumphosphate is used to control pH, and an ionicstrength of at least 0.02 is recommended tosafeguard against possible ionic interactionswith the gel matrix. NaCl can be used for thispurpose.
* For the product that is to be lyophilized:Use volatile buffers e.g. ammonium acetate,ammonium bicarbonate or ethylenediamine acetate.---------------------------------------------------------------------------------Cleaning (Details)
===============================================* Wash through 2 column volumes of a non-ionic
detergent solution.
* Wash with 0.2 M NaOH outside the column due toswelling.
---------------------------------------------------------------------------------Colour (Details)
===============================================White.
---------------------------------------------------------------------------------Column packing (Details)
===============================================
The gel suspension should reach the temperature of column operation before packing is begun.1. Inject eluent buffer into the outlet tubing untilit passes through the bed support net.
2. Close the outlet tubing, when the dead space underthe net is properly filled.
3. Pour the entire slurry into the column in a singleoperation. (Use reservoir if necessary.)4. Open the outlet valve.
5. Start the pump or gravity flow to initiatepacking. The column should be packed at as highpressure as possible without deforming beads.The pressure should not be increased beyond 9.6kPa (value for 2.6 x 30 cm column in aqueousbuffer at room temperature. It may vary dependingon eluent viscosity. With wider columns, slightlyreduced maximum operating pressures must be used.)6. Remove the reservoir when all the gel hassedimented into the column.7. Insert the adaptor.
8. Pack at the maximum operating pressure of the gel.9. Adjust the flow adaptor to the surface of thegel bed when the gel is thoroughly packed into thecolumn.
---------------------------------------------------------------------------------Composition of the gel matrix (Details)
===============================================Dextran, cross-linked with epichlorohydrin.
Sephadex contains a small number of carboxyl groups, which at low ionic strength cause interaction between chargedsolutes and the matrix.
---------------------------------------------------------------------------------Density (Details)
===============================================We do not measure density.
---------------------------------------------------------------------------------Equilibration (Details)
===============================================1. Pass 2 to 3 column volumes of the aqueous bufferto be used in the separation.
2. Readjust the flow adaptor to the surface of thebed.
---------------------------------------------------------------------------------Exclusion limit (Details)
===============================================Dextrans95 kD
Buffer solution: Phosphate buffer 0.05 M- NaCl 0.15 M - NaN3 0.01% pH 7.0.-----Globular proteins210 kD
Buffer solution: Phosphate buffer 0.05 M- NaCl 0.15 M - NaN3 0.01% pH 7.0.
---------------------------------------------------------------------------------Expected shelf life (Details)
===============================================8 years.
---------------------------------------------------------------------------------Form as supplied (Details)
===============================================Dry powder.
---------------------------------------------------------------------------------
Fractionation range (Details)
=============================================== Dextrans1 kD - 100 kD
Buffer solution: Phosphate buffer 0.05 M- NaCl 0.15 M - NaN3 0.01% pH 7.0.-----Globular proteins2 kD - 120 kD
Buffer solution: Phosphate buffer 0.05 M- NaCl 0.15 M - NaN3 0.01% pH 7.0.-----Peptides2 kD - 120 kD
Buffer solution: Info not available.
---------------------------------------------------------------------------------Major separation mechanism (Details)
=============================================== According to size.---------------------------------------------------------------------------------Max. linear flow rate (Details)
===============================================50 cm/h.
Values may vary depending on column packing and eluent viscosity.---------------------------------------------------------------------------------Max. operating pressure (Details)
===============================================9.6 kPa
Value for 2.6 x 30 cm column, in aqueous buffer at room temperature. Values may vary depending on column packing andeluent viscosity.
---------------------------------------------------------------------------------Max. volumetric flow rate (Details)
===============================================4.2 ml/min
Value for 2.6 x 30 cm column, in aqueous buffer at room temperature. Values may vary depending on column packing andeluent viscosity.
---------------------------------------------------------------------------------Optimisation (Details)===============================================
* Parameters to change are:A. Sample volume.
- A smaller sample volume gives betterresolution.B. Flow rate.
¤ Lower flow rate gives better resolution forhigh molecular weight components.
¤ The opposite may be true for small componentssince they diffuse more quickly.
- The longer the separation time is, the widerthe sample zones become.C. Column length.
¤ The resolution of two separated zonesincreases as the square root of the columnlength.
¤ The effective bed height can be increased by:- Recycling.
- Coupling two columns in series.
* Buffer choice and pH are normally of minorimportance.
---------------------------------------------------------------------------------pH stability; operational (Details)=============================================== 2 - 10---------------------------------------------------------------------------------Range of dry bead size (Details)
=============================================== 40 - 120 µm
---------------------------------------------------------------------------------Range of wet bead size (Details)=============================================== 100 µm - 310 µm---------------------------------------------------------------------------------Rec. column (Details)
=============================================== Long, narrow column e.g. C 16, 26 / 40 - 100 cmXK 16, 26 / 40 - 100 cm
* The length of the column is decided by theresolution required, (the resolution of twoseparated zones in gel filtration increases as thesquare root of column length) and the diameter bythe sample size.
* The effective bed height can be increased byrecycling or using columns coupled in series.
---------------------------------------------------------------------------------Rec. linear flow rate (Details)=============================================== 2 - 5 cm/h---------------------------------------------------------------------------------Sample volume fractionation (Details)
=============================================== 1 - 5 % of bed volume.---------------------------------------------------------------------------------Solvent regain (Details)
=============================================== Distilled water 9 - 11 ml/g.---------------------------------------------------------------------------------Storage of unused material (Details)
=============================================== 4 - 25°C, dry.---------------------------------------------------------------------------------Storage of used material (Details)
=============================================== 4 - 8°C, pH 6 - 8, do not freeze.Bacteriostatic agent (e.g. 20 % ethanol, 0.04 % sodium azide). Storage of column:1. Wash with 0.04 % sodium azide.
2. Put a stop screw into a bottom tubing of thecolumn and insert the tubing from the adaptor ina Parafilm a protected vessel (e.g. test tube)with 20 % ethanol.
---------------------------------------------------------------------------------Swelling dry gel (Details)
=============================================== Swelling time: 72 h at 20°C or 5 h at 90°C.1. Weigh out the appropriate amount of dry gel forthe required bed volume.
(Approx. bed volume: 15 - 20 ml/g dry gel.)2. Add enough buffer to equal the total volume ofthe column plus 30 % more.
3. Stir - excessive stirring should be avoided as itmay break the beads.
DO NOT USE MAGNETIC STIRRERSThe process is accelerated by using a boilingwater bath, which also serves to deaerate the
buffer.
4. Decant the supernatant (after complete swelling).5. Add back buffer to make a suspension.This should be fairly thick, (about 75 % settledgel) and de-gassed.
---------------------------------------------------------------------------------Swelling of dry material (Details)
=============================================== Dimethyl sulphoxide 45 ml/gDistilled water 15 - 20 ml/gSaline 19 ml/g
---------------------------------------------------------------------------------Temperature stability (Details)===============================================120°C.
Dry gel should not be heated to more than 120°C as it will caramelize. ---------------------------------------------------------------------------------Toxicity data (Details)
===============================================We do not have any toxicity data for our gels.They are intended for in vitro use only.
---------------------------------------------------------------------------------2-chloroethanol 30 % (Stability)
===============================================Should be OK.
---------------------------------------------------------------------------------2-mercaptoethanol 0.1 M (Stability)
===============================================OK
OK 60 min, 60°C.
---------------------------------------------------------------------------------Acetic acid 50% (Stability)
===============================================Not recommended.Degradation of the gel.
\"It seems probable that solubilization of the morehighly cross-linked gels (than Sephadex G-150) bystrong formic and acetic acids also occurs but has
escaped notice, because the most commonly appliedprocedures for examining peptide containing eluentswould not have detected the presence of thecarbohydrate.\"
---------------------------------------------------------------------------------Acetic acid 1 M (Stability)
===============================================OK
OK equilibration/elution.
---------------------------------------------------------------------------------Alkaline solutions (Stability)
===============================================OK
---------------------------------------------------------------------------------Aqueous salt solutions (Stability)=============================================== OK---------------------------------------------------------------------------------Brij 58 16 mg/ml (Stability)
=============================================== Should be OK.---------------------------------------------------------------------------------CHAPS 30 mM (Stability)=============================================== OKOK for equilibration/elution.
---------------------------------------------------------------------------------Chloroform/methanol (3:1) (Stability)
=============================================== OKOK for equilibration/elution.
---------------------------------------------------------------------------------Dextranase (Stability)=============================================== Should be avoided.---------------------------------------------------------------------------------Dimethyl sulphoxide (Stability)=============================================== OKOK for equilibration/elution.
---------------------------------------------------------------------------------Dithiothreitol 1 mM (Stability)=============================================== Should be OK.
---------------------------------------------------------------------------------Empigen BB 1 % (Stability)=============================================== Should be OK.
---------------------------------------------------------------------------------Formamide 14 M (Stability)
=============================================== Should be OK.---------------------------------------------------------------------------------Formic acid 10% (Stability)
=============================================== OKOK equilibration/elution.
---------------------------------------------------------------------------------Formic acid 50 % (Stability)=============================================== Use with care.Conflicting reports.
1. OK for equilibration/elution.2. Degradation of the gel.
\"It seems probable that solubilization of the morehighly cross-linked gels (than Sephadex G-150) bystrong formic and acetic acids also occurs but hasescaped notice, because the most commonly appliedprocedures for examining peptide, containing eluentswould not have detected the presence of the carbo-hydrate.\"
---------------------------------------------------------------------------------Glucose 0.1 M (Stability)=============================================== OKOK elution.
---------------------------------------------------------------------------------Glycine-HCl 0.1 M pH 1.0 (Stability)=============================================== OKOK elution.
---------------------------------------------------------------------------------Guanidine hydrochloride 6 M (Stability)
=============================================== OKOK equilibration/elution.
---------------------------------------------------------------------------------Hydrazine 80 % (Stability)=============================================== Should be OK.---------------------------------------------------------------------------------Hydrochloric acid 0.02 M (Stability)
=============================================== OKThe gel was well soaked in the cold room in 0.02 M HCl.
---------------------------------------------------------------------------------Methanol 50% (Stability)=============================================== OK
OK elution.
---------------------------------------------------------------------------------Organic solvents (Stability)=============================================== OK---------------------------------------------------------------------------------Oxidizing agents (Stability)
=============================================== Should be avoided.---------------------------------------------------------------------------------Piperidine 1 M (Stability)
=============================================== Should be OK.---------------------------------------------------------------------------------Propionic acid 1 M (Stability)
=============================================== OKOK equilibration/elution.
---------------------------------------------------------------------------------Sodium cholate 15 mM (Stability)=============================================== OKOK for equilibration/elution.
---------------------------------------------------------------------------------Sodium deoxycholate 16 mg/ml (Stability)=============================================== Should be OK.
---------------------------------------------------------------------------------Sodium dodecyl sulphate 3 % (Stability)=============================================== Should be OK.---------------------------------------------------------------------------------Strong acids (Stability)=============================================== Should be avoided.---------------------------------------------------------------------------------Triton X-100 30 mM (Stability)
=============================================== OKOK for equilibration/elution.
---------------------------------------------------------------------------------Urea 8 M (Stability)=============================================== OKOK equilibration/elution.
---------------------------------------------------------------------------------Weakly acidic solutions (Stability)
=============================================== OK---------------------------------------------------------------------------------
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