Harvard Apparatus Gel Filtration or Size Exclusion Chromatography Guide
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Guide to Gel Filtration or Size Exclusion Chromatography

Introduction Gel Filtration Gel Filtration also called size-exclusion chromatography can be used for protein DNA purification, buffer exchange, desalting, or for group separation in which the sample is separated in two major groups. Gel Filtration is an easy to use method for separation of molecules with different molecular sizes, using mild conditions. Gel Filtration uses the size of molecules in solution to determine separation. SpinColumns have short media packing so the samples are separated by size, the large molecules travel out of the column with the void volume the smaller...

Introduction (cont.) In Gel Filtration, maximum resolution can be obtained with sample volumes of 0.5% to 2% of the total column volume; however, up to 5% may give acceptable separation. Even larger samples volumes can be appropriate if the resolution between target protein and the impurities to be removed is high. To increase capacity, the sample can be concentrated before Gel Filtration or larger columns can be used. Sample components are eluted isocratically (single buffer, no gradient). Separation can be performed within a broad pH, ionic strength, and temperature range, and the medium...

Introduction (cont.) The choice of buffer will not affect resolution, but a low concentration of salt, between, 25 and 150 mM NaCl, should be used to reduce weak electrostatic interactions between proteins and the Gel Filtration media. The selected buffer conditions should be one that does not cause inactivation or precipitation, but maintains the biomolecules stability and target proteins activity. Proteins are large molecules and cannot enter the pores of chromatography beads, but a protein that fits into a pore of the beads, will be retained/retarded. Salt or other low molecular weight...

Size Fractionation Gel Filtration can be used for size fractionation of different sized molecules in a sample. The separation of molecules in a sample will be by molecular weight distribution. When separating by size fractionation the sample result you will obtain will contain a few components, large or small molecules, if separating a complex mixture of many components gel filtration is not a good fit. And will result in poor resolution. Size Fractionation is a good final step for purification. Sample Volume For group separation use volumes up to 30% of the total column volume. A sample...

Buffer Sample Selection Samples and Buffer Samples should be free of particulate matter, especially when working with bead size of 34µm or less. For post extraction clean-up two approaches are available, centrifugation and filtration. Filtration The sample extract mixture is passed through a filter membrane to remove the solid sample form the solute. Fresh solvent washes the sample from the filter into the collection vessel. Two to three washes can be used to prevent sample dilution. Centrifugation The sample extract mixture is centrifuges and the extract is decanted and removed. The...

Buffer Sample Selection (cont.) Buffer Selection Select a buffer that supports protein stability and activity. The buffer should maintain the buffering capacity and constant pH, 25mM-150mM NaCl will avoid nonspecific, ionic interactions. If using guanidine hydrochloride or urea to stabilize the protein, during extraction it should be included in the buffer. Detergents are useful as stabilizing agents for proteins with low aqueous solubility and will not effect separation. If using detergents to stabilize a sample, they should be present in both the sample buffer and running buffer. If a...

Selection of Media and Size Harvard Apparatus Offers Six Types Of Media In Five Different Sizes Media Selection Type Desalting Peptides Removal of Free labels from labeled macromolecules Molecular weight determination Rapid carbohydrate,& small peptide separations & desalting Purification of proteins & polypeptides Desalting Proteins & Nucleic Acids SpinColumn Specifications Description Sample Volume Elution Volume

Gel Filtration SpinColumns Gel Filtration SpinColumns G-10, G25, G50 and G-100 The Gel Filtration SpinColumns G-10, G25, G50 and G-100 are packed with Sephadex. Sephadex is a highly cross linked porous agarose particles with is covalently bonded. The media has high physical and chemical stability, due to the highly crosslinked agarose matrix. The excellent properties are determined by the dextran chains. The stability of Superdex makes it suitable for use in SpinColumns where centrifugation at moderate speed spin protocols are required. Under normal chromatography conditions nonspecific...

Spehadex G-25 Applications Sephadex G-25 Application Superfine For highest column efficiency (highest resolution), but operating pressures increase Small-scale separations 10µl to 150µl. G-25 SpinColumns to remove salts and other low molecular weight compounds from proteins with MW > 5000 and Sephadex G-10 products for proteins with MW > 700. Desalting (SEC) provides several advantages over dialysis for desalting. Dialysis requires a longer period of time and a large volume of buffer. Additionally protein activity and/or stability can be compromised during dialysis if improperly handled....

Desalting Column Applications Applications Desalting columns are used not only to remove low molecular weight contaminants such as salt, but also for buffer exchange before and after different chromatography techniques and for the rapid removal of reagents to terminate a reaction. Examples of group separations include: • Removal of phenol red from culture fluids prior to anion exchange chromatography or nucleic acid preparations Removal of unincorporated nucleotides during DNA sequencing Removal of free low molecular weight labels Termination of reactions between macromolecules and low...

P-2, P-6 and P-30 SpinColumns P-2, P-6 and P-30 SpinColumns P-2, P-6 and P-30 SpinColumns are porous polyacrylamide beads prepared by copolymerization of acrylamide and N,N'-methylene-bis-acrylamide. The SpinColumns are extremely hydrophilic and essentially free of charge, and provide efficient gel filtration of sensitive compounds. The composition and lack of soluble impurities eliminate sample contamination. The consistency of bead diameter gives high resolution separation by molecular weight. P-2, P-6 and P-30 SpinColumns is compatible with dilute organic acids, 8 M urea, 6 M...